Susan Hayward

Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation and gonadectomy

A heterologous inhibin radioimmunoassay (RIA) method has been developed which is highly specific and of sufficient sensitivity to detect inhibin in female and male rat serum. Purified bovine 31 kDa inhibin was used in the generation of the antiserum and following iodination as tracer in the assay. Parallel logit-log dose-response lines were observed between a rat ovarian extract used as standard and serial dilutions of female and male serum and testicular interstitial fluid. The within-assay variation based on index of precision was 0.049 (n = 5) and the between-assay variation (n = 4) was 9.8%. The specificity of the assay was assessed from: (a) the failure of a number of structurally related proteins (activin-A, transforming growth factor-beta (TGF-beta), Müllerian inhibitory substance) as well as inhibin subunits to crossreact (less than 0.5%) in the assay relative to bovine 31 kDa inhibin; (b) nondetectable levels of immunoactivity in the serum of gonadectomised rats; and (c) a close correlation (r = 0.96) between serum levels of in vitro biological and immunological activities from rats following pregnant mare serum gonadotropin (PMSG) stimulation. Similar initial t 1/2 values (14-15 min) of serum inhibin following gonadectomy were obtained in both sexes. This RIA method will be useful in the study of the physiology of inhibin in the female and male rat.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

The Role of Activin A and B and the Benefit of Follistatin Treatment in Renal Ischemia-Reperfusion Injury in Mice.

Background Activins, members of the TGF-&bgr; superfamily, are key drivers of inflammation and are thought to play a significant role in ischemia-reperfusion injury (IRI), a process inherent to renal transplantation that negatively impacts early and late allograft function. Follistatin (FS) is a protein that binds activin and inhibits its activity. This study examined the response of activin A and B in mice after renal IRI and the effect of exogenous FS in modulating the severity of renal injury. Methods Mice were treated with recombinant FS288 or vehicle before renal IRI surgery. Activin A, B, and FS levels in the serum and kidney, and renal injury parameters were measured at 3, 6, and 24 hours after reperfusion. Results Serum and kidney activin B levels were increased within 6 hours postrenal IRI, accompanied by renal injury—increased serum creatinine, messenger (m)RNA expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL); endothelial activation—increased E-selectin mRNA; and systemic inflammation—increased serum levels of IL-6, monocyte chemotactic protein-1 and TNF-&agr;. Further injury was potentiated by an upsurge in activin A by 24 hours, with further increases in serum creatinine, KIM-1 and NGAL mRNA expression. Follistatin treatment significantly reduced the level of serum activin B and subsequently blunted the increase in activin A. Renoprotection was evident with the attenuated rise in serum creatinine, KIM-1 and NGAL expression, tubular injury score, renal cell apoptosis, and serum IL-6 and monocyte chemotactic protein-1 levels. Conclusions We propose that activin B initiates and activin A potentiates renal injury after IRI. Follistatin treatment, through binding and neutralizing the actions of activin B and subsequently activin A, reduced renal IRI by minimizing endothelial cell activation and dampening the systemic inflammatory response. These data support the potential clinical application of FS treatment to limit IRI during renal transplantation.

Testicular activin and follistatin levels are elevated during the course of experimental autoimmune epididymo-orchitis in mice

Experimental autoimmune epididymo-orchitis (EAEO) is a model of chronic inflammation, induced by immunisation with testicular antigens, which reproduces the pathology of some types of human infertility. Activins A and B regulate spermatogenesis and steroidogenesis, but are also pro-inflammatory, pro-fibrotic cytokines. Expression of the activins and their endogenous antagonists, inhibin and follistatin, was examined in murine EAEO. Adult untreated and adjuvant-treated control mice showed no pathology. All mice immunised with testis antigens developed EAEO by 50 days, characterised by loss of germ cells, immune cell infiltration and fibrosis in the testis, similar to biopsies from human inflamed testis. An increase of total CD45+ leukocytes, comprising CD3+ T cells, CD4 + CD8− and CD4 + CD25+ T cells, and a novel population of CD4 + CD8+ double positive T cells was also detected in EAEO testes. This was accompanied by increased expression of TNF, MCP-1 and IL-10. Activin A and B and follistatin protein levels were elevated in EAEO testes, with peak activin expression during the active phase of the disease, whereas mRNA expression of the inhibin B subunits (Inha and Inhbb) and activin receptor subunits (Acvr1b and Acvr2b) were downregulated. These data suggest that activin–follistatin regulation may play a role during the development of EAEO.

Follistatin Reduces Serum Activin and Attenuates Renal Ischemia-Reperfusion Injury in Mice.: Abstract# C1686

C1687 5,7-Dihydroxy-3,4,6-Trimethoxyflavone Protects Kidneys From Ischemia-Reperfusion Injury in Mice. H. Jang, E. Jeong, S. Kim, J. Lee, K. Kang, D. Eom, D. Han. Surgery, University of Ulsan Gangneung Asan Hospital, Gangneung, Gangwon, Korea, Republic of; Anesthesia, University of Ulsan Gangneung Asan Hospital, Gangneung, Gangwon, Korea, Republic of; Anesthesia, University of Ulsan Gangneung Asan Hospital, Gangneung, Gangwon, Korea, Republic of; Natural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung, Gangneung, Gangwon, Korea, Republic of; Natural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung, Gangneung, Gangwon, Korea, Republic of; Pathology, University of Ulsan Gangneung Asan Hospital, Gangneung, Gangwon, Korea, Republic of; Surgery, University of Ulsan Asan Medical Center, Seoul, Korea, Republic of. 5,7-dihydroxy-3,4,6-trimethoxyfl avone (Eupatilin), a pharmacologically active fl avone derived from the Artemisia plant species, has been reported to have antioxidant and anti-infl ammatory activities. Ischemia-reperfusion injury (IRI) remains a major problem in renal transplantation, and the infl ammatory response to IRI exacerbates the resultant renalinjury. In the present study, we investigated whether eupatilin exhibits renoprotective activities against ischemia/reperfusion-induced acute kidney injury (AKI) in mice. Renal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 min followed by reperfusion for 48 hr. Eupatilin (10 mg/kg, p.o.) was administered 4 days before IRI. Treatment with eupatilin signifi cantly decreased blood urea nitrogen, serum creatinine levels, as well as kidney tubular injury. [fi gure 1] Western blotting indicated that eupatilin further signifi cantly increased the level of HSP70 at 48 h after IRI [fi gure 2], attenuated the level of caspase 3. These fi ndings suggest that eupatilin is a promising therapeutic agent against acute ischemia-induced renal damage and that its renoprotective effects may be mediated in part by increased HSP70. Abstract# C1688 Transgenic Overexpression of Cardiac-Specifi c Vascular Endothelial Growth Factor B Exacerbates Ischemia Reperfusion Injury in Rat Cardiac Grafts. A. Raissadati,1 R. Tuuminen,1 A. Dashkevich,1 R. Krebs,1 R. Arnaudova,1 E. Rouvinen,1 K. Alitalo,2 A. Nykanen,1 K. Lemstrom.1 1Transplantation Laboratory, Haartman Institute, University of Helsinki and Cardiac Surgery, Heart and Lung Center, Helsinki University Central Hospital, Helsinki, Finland, Helsinki, Finland; 2Molecular/Cancer Biology Program, Institute for Molecular Medicine Finland and Helsinki University Central Hospital, Research Programs Unit, Biomedicum Helsinki, University of Helsinki, Finland, Helsinki, Finland. Purpose: Examine the effects of long-term cardiac-specifi c VEGF-B overexpression on ischemia-reperfusion injury (IRI) in rat heart transplants. Methods: Intra-abdominal heterotopic heart transplantations were performed from transgenic VEGF-B overexpressing Wistar to Wistar Furth rats. Grafts were exposed to 2h cold and 1h warm ischemia prior to reperfusion and recovered after 6h. Recipient serum cTnT was measured and in situ TUNEL assay and Oily red-O (ORO) staining of cardiac graft cross-sections performed. Intragraft infl ammation was examined with immunohistological stainings. Bax and Bcl-genes and a variety of metabolic genes were measured with RT-PCR. Cardiac and cellular dimensions were measured with computer-assisted imaging. Results: Recipient serum cTNT was signifi cantly higher in the VEGF-B group (Figure 1A). There were no differences in the amount of TUNEL+ cells, ratio of Bax to Bcl2 mRNA (Figure 1B) or quantity of infl ammatory cells. There was a signifi cant increase in CMC size, Cytochrome C oxidase subunit I (COX I) and CD39 mRNA in the VEGF-B group, but no differences in cardiac cross-sectional area (Figure 1C-D). Conclusions: Chronic cardiac VEGF-B over-expression exacerbates IRI. Increased CMC size, COX I and CD39 mRNA levels in the VEGF-B grafts suggest a metabolic difference between the groups. Previous studies have shown that VEGF-B increases fatty acid intake into CMCs and decreases insulin sensitivity. We hypothesize that chronic cardiac VEGF-B over-expression increases CMC energy demand and impairs cellular glucose utilization, disrupting the CMC metabolic switch to anaerobic glycolysis during graft ischemia time and rendering them more susceptible to an ischemic insult.