Relaxin Gene Research Articles

822: Genetic susceptibility to preterm birth in the Filipino population in Hawaii

822 Genetic susceptibility to preterm birth in the Filipino population in Hawaii Frederico Rocha, Thomas Slavin, Dongmei Li, Sandra Yamamoto, Gillian Bryant-Greenwood John A. Burns School of Medicine University of Hawaii, Department of Obstetrics, Gynecology and Women’s Health, Honolulu, HI, John A. Burns School of Medicine University of Hawaii, Department of Pediatrics, Honolulu, HI, John A. Burns School of Medicine University of Hawaii, Department of Public Health Sciences, Honolulu, HI OBJECTIVE: Filipinos have the highest preterm birth rate in the State of Hawaii. Relaxin (RLN) is a systemic hormone (sRLN) produced by the corpus luteum but is also produced by the decidua (dRLN) as a local hormone. Elevated maternal sRLN has been associated with increased risk of preterm labor (PTL) and elevated dRLN associated with preterm premature rupture of membranes (PPROM). We have sought associations of two SNPs in the RLN H2 gene promoter in Filipino patients with PTL or PPROM, as well as with their expression of cytoplasmic dRLN protein. STUDY DESIGN: Filipino patients (n 60) who delivered at 34-36 weeks gestation were subdivided into PTL (n 20) and PPROM (n 20) and compared to controls who delivered at term (n 20). Maternal DNA was used for identification by specific Taqman RT-PCR of SNPs: rs 472076 and rs 3758239. Formalin fixed sections of parietal decidua from the same patients were immunostained with a Mab to RLN H2 (Abcam) and the ImmPRESS kit (Vector Labs). All slides were blinded for multispectral imaging (Nuance) and quantitated by the Inform software. Results were expressed as mean SD and significance set at p 0.05. Kruskal-Wallis nonparametric ANOVA was used to compare differences in SNP expression and multiple comparisons were determined by the Bonferroni-Dunn’s test. RESULTS: SNP rs4742076 was significantly (p 0.0012) associated with PPROM compared to controls. The PPROM patients also had significantly more (p 0.001) dRLN expressed compared to controls. SNP rs375839 was significantly associated (p 0.0025) with both PPROM and PTL compared to the controls. However, dRLN was only significantly increased (p 0.001) in the PPROM patients. CONCLUSION: SNP rs4742076 in the RLN gene promoter and increased dRLN protein were associated with PPROM in Filipino patients in Hawaii. SNP rs3758239 was associated with both PPROM and PTL, but dRLN was only increased in the PPROM patients and not in PTL compared to the term controls. Supported by grant NIHNCRR(U54RR026136) NIHMD(U54MD007584).

882 Adenovirus mediated relaxin gene therapy improves diastolic function of hypertensive rats by activating phosphorylation of phospholamban

882 Adenovirus mediated relaxin gene therapy improves diastolic function of hypertensive rats by activating phosphorylation of phospholamban

Up-Regulating Relaxin Expression by G-Quadruplex Interactive Ligand to Achieve Antifibrotic Action

Myocardial fibrosis is a key pathological change in a variety of heart diseases contributing to the development of heart failure, arrhythmias, and sudden death. Recent studies have shown that relaxin prevents and reverses cardiac fibrosis. Endogenous expression of relaxin was elevated in the setting of heart disease; the extent of such up-regulation, however, is insufficient to exert compensatory actions, and the mechanism regulating relaxin expression is poorly defined. In the rat relaxin-1 (RLN1, Chr1) gene promoter region we found presence of repeated guanine (G)-rich sequences, which allowed formation and stabilization of G-quadruplexes with the addition of a G-quadruplex interactive ligand berberine. The G-rich sequences and the G-quadruplexes were localized adjacent to the binding motif of signal transducer and activator of transcription (STAT)3, which negatively regulates relaxin expression. Thus, we hypothesized that the formation and stabilization of G-quadruplexes by berberine could influence relaxin expression. We found that berberine-induced formation of G-quadruplexes did increase relaxin gene expression measured at mRNA and protein levels. Formation of G-quadruplexes significantly reduced STAT3 binding to the promoter of relaxin gene. This was associated with consequent increase in the binding of RNA polymerase II and STAT5a to relaxin gene promoter. In cardiac fibroblasts and rats treated with angiotensin II, berberine was found to suppress fibroblast activation, collagen synthesis, and extent of cardiac fibrosis through up-regulating relaxin. The antifibrotic action of berberine in vitro and in vivo was similar to that by exogenous relaxin. Our findings document a novel therapeutic strategy for fibrosis through up-regulating expression of endogenous relaxin.

Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

The Formation and Stabilization of a Novel G-Quadruplex in the 5′-Flanking Region of the Relaxin Gene

It has been reported that binding of STAT3 protein to the 5′-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes.

Gene Duplication and Positive Selection Explains Unusual Physiological Roles of the Relaxin Gene in the European Rabbit

The relaxin gene family is a group of genes involved in different physiological roles, most of them related to reproduction. In vertebrates the genes in this family are located in three separate chromosomal locations, and have been called relaxin family locus (RFL) A, B, and C. Among mammals the RFLA and RFLC are the most conserved as no gene copy-number variation has been observed thus far. The RFLB locus is also conserved on most mammals other than primates, where there are several gene gains and losses. Interestingly, the relaxin gene found on the RFLB locus in the European rabbit has acquired a novel role. In addition to the classical reproductive roles, this gene is expressed in tracheobronchial epithelial cells and its expression has been linked to squamous differentiation. We reconstructed the evolutionary history of the European rabbit RFLB locus using the tools of comparative genomics and molecular evolution. We found that the European rabbit possess a RFLB locus which is unique among mammals in that there are five tandemly arranged relaxin gene copies, which contrast with the single relaxin copy gene found in most mammals. In addition we also found that the ancestral pre-duplication gene was subject to the action of positive selection, and several amino acid sites were identified under the action of natural selection including the sites B12 and B13 which are part of the receptor recognition and binding site.

Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models

Monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA4) are a novel form of cancer immunotherapy. While preclinical studies in mouse tumor models have shown anti-tumor efficacy of anti-CTLA4 injection or expression, anti-CTLA4 treatment in patients with advanced cancers had disappointing therapeutic benefit. These discrepancies have to be addressed in more adequate pre-clinical models. We employed two tumor models. The first model is based on C57Bl/6 mice and syngeneic TC-1 tumors expressing HPV16 E6/E7. In this model, the HPV antigens are neo-antigens, against which no central tolerance exists. The second model involves mice transgenic for the proto-oncogen neu and syngeneic mouse mammary carcinoma (MMC) cells. In this model tolerance to Neu involves both central and peripheral mechanisms. Anti-CTLA4 delivery as a protein or expression from gene-modified tumor cells were therapeutically efficacious in the non-tolerized TC-1 tumor model, but had no effect in the MMC-model. We also used the two tumor models to test an immuno-gene therapy approach for anti-CTLA4. Recently, we used an approach based on hematopoietic stem cells (HSC) to deliver the relaxin gene to tumors and showed that this approach facilitates pre-existing anti-tumor T-cells to control tumor growth in the MMC tumor model. However, unexpectedly, when used for anti-CTLA4 gene delivery in this study, the HSC-based approach was therapeutically detrimental in both the TC-1 and MMC models. Anti-CTLA4 expression in these models resulted in an increase in the number of intratumoral CD1d+ NKT cells and in the expression of TGF-β1. At the same time, levels of pro-inflammatory cytokines and chemokines, which potentially can support anti-tumor T-cell responses, were lower in tumors of mice that received anti-CTLA4-HSC therapy. The differences in outcomes between the tolerized and non-tolerized models also provide a potential explanation for the low efficacy of CTLA4 blockage approaches in cancer immunotherapy trials.

Localization of diversified relaxin gene transcripts in the brain of eels

Relaxin 3 (RLN3) is a newly-discovered member of the insulin superfamily. We isolated three RLN3-like cDNAs from the brain of the Japanese eel (Anguilla japonica). The deduced amino acid sequences of the RLN3-like cDNAs contained the two-chain structure common to relaxin including a RXXXRXXI/V motif in the B-chain. Phylogenetic analysis assigned the two prepropeptides into teleost/mammalian RLN3 group, which are a pair of duplicates generated by the teleost-specific third-round whole genome duplication, and the other one into teleost RLN group. Therefore, they have been named eel rln3a, rln3b and rln. rln3a transcripts were abundant in the middle-posterior region of the brain and detected at lower levels in the gills, head kidney and kidney. rln3b transcripts were also detected in the middle-posterior region of the brain, but the expression levels were lower than those of rln3a. Low levels of rln transcripts were detected in all brain areas, pituitary, digestive tract and gonad. Quantitative PCR analysis did not detect differences in expression of any rln3 or rln gene between freshwater- and seawater-acclimated eels. In situ hybridization showed that rln3a was expressed in neurons of the lateral lemniscus of the midbrain and of the griseum centrale (GC) of the hindbrain, while low amounts of rln transcripts were found in neurons of the periventricular nucleus of the posterior tuberculum of the diencephalon and the GC. These results suggest that the multiple RLN3-like peptides may play regulatory roles in the brain of euryhaline fish.

Endometrial LGR7 expression during menstrual cycle

In a prospective observational study, 50 healthy patients aged 18-39 years, with regular ovulatory cycle and normal hormone levels, underwent endometrial biopsy in the proliferative and secretory phase of the menstrual cycle for semiquantitative reverse-transcription polymerase chain reaction analysis of mRNA for LGR7, the classic relaxin receptor. LGR7 is constitutively expressed in human endometrium, and an increased LGR7 immunostaining is demonstrated in the secretory phase, confirming the involvement of relaxin in the physiology of endometrium and suggesting its role in implantation.

Evolution of the Relaxin/Insulin-like Gene Family in Placental Mammals: Implications for Its Early Evolution

The relaxin (RLN) and insulin-like (INSL) gene family is a group of genes involved in a variety of physiological roles that includes bone formation, testicular descent, trophoblast development, and cell differentiation. This family appears to have expanded in vertebrates relative to non-vertebrate chordates, but the relative contribution of whole genome duplications (WGDs) and tandem duplications to the observed diversity of genes is still an open question. Results from our comparative analyses favor a model of divergence post vertebrate WGDs in which a single-copy progenitor found in the last common ancestor of vertebrates experienced two rounds of WGDs before the functional differentiation that gave rise to the RLN and INSL genes. One of the resulting paralogs was subsequently lost, resulting in three proto-RLN/INSL genes on three separate chromosomes. Subsequent rounds of tandem gene duplication and divergence originated the set of paralogs found on a given cluster in extant vertebrates. Our study supports the hypothesis that differentiation of the RLN and INSL genes took place independently in each RLN/INSL cluster after the two WGDs during the evolutionary history of vertebrates. In addition, we show that INSL4 represents a relatively old gene that has been apparently lost independently in all Euarchontoglires other than apes and Old World monkeys, and that RLN2 derives from an ape-specific duplication.

Relaxin family peptide systems and the central nervous system.

Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.

Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin

As a hallmark of heart disease, cardiac fibrosis contributes to the development of heart failure and arrhythmias and forms a key therapeutic target. There is a major unmet need for selective, potent, and safe antifibrotic drugs. Earlier studies revealed a cardiac fibrosis phenotype in relaxin-1-deficient mice. Recent studies in several rodent models of cardiac fibrosis have documented reversal of fibrosis by treatment with relaxin peptide or virally mediated relaxin gene delivery. In mice with surgically induced transmural myocardial infarction, relaxin therapy inhibited scar density. In these studies, however, functional benefits achieved by relaxin therapy were limited or less explored. Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal by relaxin and the usefulness of relaxin in antiarrhythmic or stem cell-based therapy.

Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis

BackgroundIn recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in Danio rerio and in situ hybridization used to assess the site-specific expression of the insulin 3-like gene in D. rerio testis.ResultsUp to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human RLN3, which we call rln3a and rln3b, an orthologue of human RLN2, rln, two paralogous copies of human INSL5, insl5a and insl5b, and an orthologue of human INSL3, insl3. Molecular evolutionary analyses indicated that: rln3a, rln3b and rln are under strong evolutionary constraint, that insl3 has been subject to moderate rates of sequence evolution with two amino acids in insl3/INSL3 showing evidence of positively selection, and that insl5b exhibits a higher rate of sequence evolution than its paralogue insl5a suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in D. rerio indicated that rln3a and rln3b are expressed in brain, insl3 is highly expressed in gonads, and that there was low expression of both insl5 genes in adult zebrafish. Finally, in situ hybridization of insl3 in D. rerio testes showed highly specific hybridization to interstitial Leydig cells.ConclusionsContrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) rln3 exhibits a similar evolution and expression pattern to mammalian RLN3, 2) insl3 has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) insl5 genes are highly represented and have a relatively high rate of sequence evolution in teleost genomes, but they exhibited only low levels of expression in adult zebrafish, 4) rln is evolving under very different selective constraints from mammalian RLN. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.

Relaxin-3 and RXFP3 expression, and steroidogenic actions in the ovary of teleost fish

Relaxins are peptides similar in secondary structure to insulins. In teleost genomes, five or six relaxin genes have been identified. Two relaxins group closely with mammalian relaxin-3 on phylogenetic analysis and are named relaxin-3a and b. We refer to the remainder as relaxins c to f. Ovarian expression of relaxin-3a, d and f genes, and the relaxin-3 receptor gene Rxfp3, was studied in Danio rerio using RT-PCR. Immunohistochemistry was used to determine the distribution of relaxin-3 peptides and RXFP3 in the ovary of Fundulus heteroclitus (killifish). Thirdly, enzyme immunoassays and ovarian follicular culture were used to determine the effect of treatment with human recombinant relaxin-3 on the production of 17β-estradiol and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in killifish ovarian follicles. Relaxin-3a, d, f, and Rxfp3 genes were expressed regardless of sex or reproductive condition. Relaxin-3 immunostaining was present in mid to late follicular stages within cortical alveoli of the oocyte cytopasm, whereas receptor staining was localized to follicular cells. Treatment with relaxin-3 enhanced 17β-estradiol production in early and late maturing follicles, but did not have an effect in vitellogenic follicles. Relaxin-3 appeared to suppress the release of MIS production. This suggests that relaxin peptides may be involved with estradiol-dependant events in follicular development.

Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to β 2-adrenergic receptor (β 2AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in β 2AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.

Relaxin-Expressing, Fiber Chimeric Oncolytic Adenovirus Prolongs Survival of Tumor-Bearing Mice

Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.

228. Relaxin Expression from Tumor-Targeting Adenoviruses and Its Intratumoral Spread, Apoptosis Induction, and Efficacy

Background The use of oncolytic adenoviruses as cancer gene therapy is limited by their uneven penetration and distribution in tumors. We investigated whether the expression of the cell matrix-degradative protein relaxin by adenovirus could improve adenovirus distribution and penetration in tumors. Methods We generated relaxin-expressing, replication-incompetent (dl-lacZ-RLX) and -competent (Ad-deltaE1B-RLX) adenoviruses by inserting a relaxin gene into the E3 adenoviral region. Controls were parental adenoviruses (dl-lacZ and Ad-deltaE1B) and phosphate-buffered saline (PBS) (vehicle). Replication-incompetent viruses, which do not lyse cells, were used to assess transduction efficiency. Viral spread in tumor spheroids, made by dissecting tumor tissue into homogeneous fragments, was assessed by reporter gene (i.e., lacZ) expression. Tumor growth inhibition was assessed by injecting adenoviruses into xenograft tumors in athymic mice (n = 8 or 9). Overall survival was assessed by the Kaplan-Meier method. Extracellular matrix was examined with Masson's trichrome staining. Therapeutic efficacy was evaluated by assessing spontaneous pulmonary metastasis in the B16BL6 melanoma mouse model and growth inhibition of orthotopically implanted hepatoma (n = 4-6). All statistical tests were two-sided. Results In tumor spheroids and established solid tumors in vivo, transduction with dl-lacZ-RLX, compared with parental virus or vehicle, elicited higher transduction efficiency and viral spread throughout the tumor mass. Infection with Ad-deltaE1B-RLX, compared with parental virus, elicited greater viral persistence and spread, leading to increased survival (e.g., 100%, 95% confidence interval [CI] = 63.1% to 100%, for C33A tumor-bearing mice treated with Ad-deltaE1B-RLX, and 50%, 95% CI = 15.7% to 84.3%, for C33A tumor-bearing mice treated with Ad-deltaE1B). Infection with Ad-deltaE1B-RLX substantially decreased the collagen content of tumor tissue but not of adjacent normal tissue, compared with noninfected tissues. Intratumoral injection of Ad-deltaE1B-RLX inhibited the formation of lung metastases in mice (PBS = 268 mg of metastatic tumor per mouse and Ad-deltaE1B-RLX = 10 mg; difference = 258 mg, 95% CI = 94 to 426; P = .003, Mann-Whitney test). Systemic treatment with Ad-deltaE1B-RLX completely inhibited the growth of Hep1 hepatocellular carcinomas (PBS = 20.2 mg of tumor per mouse and Ad-deltaE1B-RLX = 0 mg; difference = 20.2 mg, 95% CI = 3.7 to 36.7; P = .004, Mann-Whitney test). Conclusion Extracellular matrix degradation by relaxin expressed by adenoviruses increased viral distribution and tumor penetration, inhibited tumor growth and metastasis, and increased survival of mice.

Relaxin Expression From Tumor-Targeting Adenoviruses and Its Intratumoral Spread, Apoptosis Induction, and Efficacy

The use of oncolytic adenoviruses as cancer gene therapy is limited by their uneven penetration and distribution in tumors. We investigated whether the expression of the cell matrix-degradative protein relaxin by adenovirus could improve adenovirus distribution and penetration in tumors. We generated relaxin-expressing, replication-incompetent (dl-lacZ-RLX) and -competent (Ad-deltaE1B-RLX) adenoviruses by inserting a relaxin gene into the E3 adenoviral region. Controls were parental adenoviruses (dl-lacZ and Ad-deltaE1B) and phosphate-buffered saline (PBS) (vehicle). Replication-incompetent viruses, which do not lyse cells, were used to assess transduction efficiency. Viral spread in tumor spheroids, made by dissecting tumor tissue into homogeneous fragments, was assessed by reporter gene (i.e., lacZ) expression. Tumor growth inhibition was assessed by injecting adenoviruses into xenograft tumors in athymic mice (n = 8 or 9). Overall survival was assessed by the Kaplan-Meier method. Extracellular matrix was examined with Masson's trichrome staining. Therapeutic efficacy was evaluated by assessing spontaneous pulmonary metastasis in the B16BL6 melanoma mouse model and growth inhibition of orthotopically implanted hepatoma (n = 4-6). All statistical tests were two-sided. In tumor spheroids and established solid tumors in vivo, transduction with dl-lacZ-RLX, compared with parental virus or vehicle, elicited higher transduction efficiency and viral spread throughout the tumor mass. Infection with Ad-deltaE1B-RLX, compared with parental virus, elicited greater viral persistence and spread, leading to increased survival (e.g., 100%, 95% confidence interval [CI] = 63.1% to 100%, for C33A tumor-bearing mice treated with Ad-deltaE1B-RLX, and 50%, 95% CI = 15.7% to 84.3%, for C33A tumor-bearing mice treated with Ad-deltaE1B). Infection with Ad-deltaE1B-RLX substantially decreased the collagen content of tumor tissue but not of adjacent normal tissue, compared with noninfected tissues. Intratumoral injection of Ad-deltaE1B-RLX inhibited the formation of lung metastases in mice (PBS = 268 mg of metastatic tumor per mouse and Ad-deltaE1B-RLX = 10 mg; difference = 258 mg, 95% CI = 94 to 426; P = .003, Mann-Whitney test). Systemic treatment with Ad-deltaE1B-RLX completely inhibited the growth of Hep1 hepatocellular carcinomas (PBS = 20.2 mg of tumor per mouse and Ad-deltaE1B-RLX = 0 mg; difference = 20.2 mg, 95% CI = 3.7 to 36.7; P = .004, Mann-Whitney test). Extracellular matrix degradation by relaxin expressed by adenoviruses increased viral distribution and tumor penetration, inhibited tumor growth and metastasis, and increased survival of mice.

The Effects of Relaxin and Estrogen Deficiency on Collagen Deposition and Hypertrophy of Nonreproductive Organs

In this study, we determined the effects of relaxin and estrogen deficiency and estrogen replacement therapy (ERT) on the cardiac, renal, and pulmonary phenotypes of female relaxin gene knockout (Rln1-/-) and age-matched wild-type (Rln1+/+) mice. One-month-old Rln1+/+ and Rln1-/- mice were bilaterally ovariectomized or sham-operated and aged until 9 or 12 months. A subgroup of ovariectomized mice received ERT from 9 to 12 months of age. At the appropriate time points, heart, kidney, and lung tissues from these mice were collected and analyzed for changes in organ fibrosis, hypertrophy, and airway thickening. Neither ovariectomy nor ERT had any effect on cardiac or renal collagen concentration in all groups studied. In contrast, total lung collagen concentration and airway subepithelial collagen deposition were significantly increased in ovariectomized Rln1+/+ mice (P<0.05 vs. sham) and to a greater extent in ovariectomized Rln1-/- mice (P<0.01 vs. sham). Ovariectomy of Rln1+/+ mice also led to a significant increase in airway smooth muscle (SM) (lung) thickening, which was further exaggerated in Rln1-/- mice. Cardiac hypertrophy, evidenced by increased heart weight and expression of hypertrophy-related genes (all P<0.05 vs. sham) was only observed in Rln1-/- mice. These findings demonstrated an increased pathology in mice that were deficient of both relaxin and estrogen. ERT significantly decreased airway fibrosis, airway SM thickening, and cardiac hypertrophy when administered to ovariectomized Rln1-/- mice (all P<0.05 vs. ovariectomy alone). These findings suggest that relaxin and estrogen appear to play protective roles against airway fibrosis, airway SM thickening, and cardiac hypertrophy in female mice.

Does a “500 million‐year‐old hormone” disprove Darwin?

In a paper published by the FASEB Journal in 1999, Danielle Georges and Christian Schwabe described gene sequences from the tunicate Ciona intestinalis that were indistinguishable from porcine relaxin (1). Newer data contradict that finding: recently published analyses of the C. intestinalis genome do not confirm the presence of relaxin sequences (2). What might seem like a narrow issue of scientific fact has broader implications. To the delight of creationists and fans of intelligent design, the presence of similar relaxin sequences in pigs and sea squirts–species separated by 500 million years–has been used to cast genomic doubt on Darwinian evolution. Indeed, Schwabe has repeatedly cited the tunicate relaxin data, and the nearly identical relaxin sequences he has identified in whales, (3) not only to support his own “Genomic Potential Hypothesis” but also to refute modern evolutionary biology (4). Schwabe’s hypothesis would replace Darwin’s theory of common descent with the view of the independent origin of all species, thereby making the origin of life and the origin of species one and the same problem. Moreover, the “Genomic Potential Hypothesis” rejects natural selection of random variations as the mechanism that underlies speciation and adaptation. Thus, albeit Schwabe sees chemistry as the sole force generating genomes and species the independent origin of all species asserted by his “Genomic Potential Hypothesis” coincides with the creationist view of the origin of species. Since Schwabe therefore is regularly cited by creationists as an “atheistic scientist” who rejects neoDarwinism, it seems worthwhile to critique his experimental work in the context of his “Genomic Potential Hypothesis.” It’s an hypothesis that wants to have it both ways. On the one hand, Schwabe argues that the high degree of diversity found in the relaxin sequences of different species would date speciation events much earlier than phylogenies based on other data. On the other hand, he argues that the identity of tunicate, cetacean, and porcine relaxin sequences cannot be explained by Darwinian selection of random mutants. This concomitant use of sequence diversity and sequence identity to deny the theory of evolution appears to us at least curious or at worst tautologous. The 1999 The FASEB Journal paper immediately evoked critical responses summarized by an editor’s comment (5) that the conclusions rested entirely on PCR and microsequencing data, two methods quite prone to contamination. The authors rejected those concerns and replied that the experiments were carried out in two separate labs and that Georges had never worked with porcine material before (6). Not so: Georges had indeed used polyA -RNA from pregnant sow ovaries (the organ in which relaxin is produced) in work published well before The FASEB Journal paper (7). At any rate, since the genomic sequence was reported to lack introns, the cDNA could have served as a good probe for Southern blot analysis of genomic DNA, a far more robust technique (a description of Southern blot analysis appears in the Methods section of The FASEB Journal paper but such data are not presented in the Results section). Indeed, this approach would have enabled the authors to decide whether the sequence variation they observed in one tenth of the PCR products was introduced during amplification, whether it resulted from a polymorphism of the gene or if two different relaxin genes reside within the Ciona genome as the authors speculated. In point of fact, polymorphisms would contradict the “Genomic Potential Hypothesis,” because, as rigidly interpreted, the Schwabe hypothesis would predict the independent origins of individuals carrying different alleles (see below). Still, the authors adduce the fact that Ciona relaxin is more similar to an infrequent allele of porcine relaxin. They suggest an “occasional exchange” in the respective position of the porcine gene: but that of course implies a mutation. Curiously, Schwabe willingly accepts that “chance mutations” may cause inherited human diseases (8) but denies that mutations can drive evolution. In addition it should be