Activin A is a cytokine whose multiple functions have yet to be fully determined. In this study, the role of proinflammatory cytokines in regulatory control of activin A production was shown in synoviocytes and chondrocytes. Additional facets of functional inflammation-related activities of activin A were also determined. Results showed that activin A concentrations in the synovial fluid of patients with rheumatoid arthritis and gout were elevated relative to those in patients with osteoarthritis. Further studies showed that production of activin A by synoviocytes and chondrocytes in culture was stimulated by cytokines such as IL-1, transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), and IL-8, consistent with previous studies in regard to the control of activin A production in marrow stromal cells and monocytes by cytokines, glucocorticoids and retinoic acid. In addition, the relationship of activin A to IL-6-induced biological activities was investigated. Three major IL-6 activities involved in inflammatory responses were found to be suppressed by activin A. In a dose-dependent manner, activin A efficiently suppressed IL-6-induced proliferation of 7TD1 B lymphoid cells, phagocytic activity of monocytic M1 cells, and fibrinogen production in HepG2. Therefore, it is likely that activin A serves as a suppressor for IL-6, dampening inflammatory responses, and has the potential to perform some previously unrecognized roles in inflammation.

Adiponectin is a well-known adipokine with insulin sensitizing and anti-inflammatory actions. Low circulating levels of adiponectin are associated with a proinflammatory milieu leading to the development of cardiovascular and metabolic disorders. We have previously shown that male adiponectin deficient (adipo-/-) mice have lower baseline blood pressures but are more susceptible to prohypertensive effect of Angiotensin II (ANGII). In addition to being a vasoconstrictor, ANGII is also known to stimulate proinflammatory cytokine expression in cardiovascular & renal tissue. However, low dose infusion of ANGII for 14 days did not increase plasma TNFα or IL6 appreciably in the adipo -/- mice. The present study evaluates the expression of these proinflammatory cytokines in renal tissue. Briefly, male wild type C57Bl/6J and adiponectin deficient (adipo-/-) mice (24-28wk, ~30g) were implanted with osmotic pumps containing Angiotensin II (ANGII, 800ng/Kg body weight/min) or Saline. At the end of 14 days infusion, the mice were euthanized to obtain blood and tissue samples. The kidneys were snap frozen in liquid nitrogen and the total RNA was extracted using RNA mini kit (Qiagen). Quantitative real-time PCR was performed with Eppendorf Realplex 4 mastercycler and SYBR Green ROX Mastermix. TNFα mRNA expression normalized to GAPDH was similar in adipo-/- and C57Bl/6J mice. TNFα mRNA expression levels were not increased by ANGII infusion. IL6 expression was undetectable in all groups (n=3 animals/group). We also carried out additional microarray analysis of markers of endothelial activation and adhesion molecules. Preliminary data show that ANGII increases expression of E-Selectin, VCAM1, Collagen1a1 and eNOS by two fold in the C57BL/6J mice. The saline treated adipo-/- had lower expression of eNOS, VCAM-1 and Collagen1a1. Interestingly, ANGII infusion increased expression of E-Selectin, VCAM1 and Collagen 1a1 in these mice but eNOS expression was unchanged. The data suggest that chronic ANGII infusion did not change renal expression of cytokines appreciably but downstream markers of inflammation and fibrosis were increased.

Circulating vasoactive peptide Angiotensin II (ANGII) has a well-known role in the development of hypertension and other cardiovascular diseases. In addition it has significant proinflammatory actions in the vascular wall inducing the production of reactive oxygen species, inflammatory cytokines and adhesion molecules. It has been previously shown that estrogen in female mice protects against ANGII mediated hypertension and against proinflammatory effects. It is not clear if the protective effects of estrogen extend to adiponectin deficient mice. Adiponectin is one of the few peptides secreted by fat to have anti-inflammatory properties. The present study evaluates the effect of chronic ANGII infusion on expression of cytokines in female C57BL/6J and adiponectin deficient mice (adipo-/-). Female mice (24- 28wks), were implanted with osmotic pump containing either ANG II (800 ng/Kg /min) or saline. Blood samples and tissue were collected at the end of 14 days. Plasma levels of TNFalpha and IL6 were measured using enzyme linked immunoabsorbent assay. Renal tissue expression of the cytokines were quantified using real-time PCR (Eppendorf Realplex 4 mastercycler) and SYBR Green ROX mastermix. Plasma TNFα levels were similar in saline infused C57Bl/6J(11±1 pg/ml) and adiponectin deficient mice(10± 1 pg/ml). ANGII did not significantly increase TNFα in the control(14±3 pg/ml) or adipo-/- mice(13+1 pg/ml). Plasma IL6 levels were also not significantly different in the two groups. A microarray for mRNA expression of markers of endothelial activation and adhesion molecules was also performed in the renal tissue. Preliminary data show that TNFalpha mRNA expression levels were not increased by ANG II infusion and IL6 expression was undetectable. ANGII also did not alter E-Selectin,VCAM1, Collagen1a1 and eNOS expression. In conclusion, ANGII infusion did not result in a proinflammatory milieu in both female C57BL/6J and Adipo -/- mice.

Adiponectin is one of the few peptides secreted by fat known to have anti-inflammatory properties. Obesity in humans is associated with low levels of circulating adiponectin. Adiponectin deficiency could potentially lead to a proinflammatory milieu, increasing susceptibility to progression of cardiovascular diseases and other inflammatory conditions. Previous studies from our lab have shown that adiponectin deficiency in mice did not lead to statistically significant increases in circulating levels of proinflammatory cytokines under basal conditions or in response to low dose infusion of Angiotensin II (ANGII). The present study evaluates the effect of chronic ANGII infusion on expression of cytokines in the livers of female C57BL/6J and adiponectin deficient mice (Adipo -/-). Female mice (24-28wks), were implanted with osmotic pump containing either ANG II (800 ng/Kg /min) or saline. Blood samples and tissue were collected at the end of 14 days. Liver tissue expression of the cytokines were quantified using real-time PCR (BioRad CFX 96 thermocycler) and SYBR Green mastermix. Data was normalized to the expression of GAPDH. Expression of cytokines IL6, IL8, IL17a was undetectable in the liver tissue of C57BL/6J and Adiponectin deficient mice. Angiotensin II infusion did not produce detectable changes in cytokine expression in either group. Expression of TNFα, TGF β , and adhesion molecules such as E-Selectin, VCAM1, Collagen1a1 in Adipo-/- mice were comparable to that of C57BL/6J mice. ANGII infusion did not produce statistically significant changes in these biomarkers either. Finally, expression of ANGII signaling components such as Angiotensin type 1 receptor (AT 1 ), NFκB and PPARγ were robustly expressed in both C57BL/6J and adiponectin deficient mice. In conclusion, chronic infusion of ANGII in Adiponectin deficiency did not result in a significant increases in expression of markers of proinflammatory milieu in the liver tissue.