Relationship between placenta growth factor 1 and vascularization, dehydroepiandrosterone sulfate to dehydroepiandrosterone conversion, or aromatase expression in patients with rheumatoid arthritis and patients with osteoarthritis.

OBJECTIVE: Proliferating pannus is in many aspects similar to placental tissue. Both fibroblast-rich tissues have high vascularity, and tissue from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA) demonstrates conversion of androgenic prehormones to downstream estrogens. We undertook this study to investigate similarities between proliferating pannus and placental tissue by focusing on angiogenic placenta growth factor 1 (PlGF-1) in patients with OA and patients with RA. METHODS: We used immunohistochemistry to study the presence of PlGF-1, its synovial distribution, and the PlGF-1-expressing synovial cell type. The relationship between PlGF-1 and conversion of the biologically inactive placental prehormone dehydroepiandrosterone sulfate (DHEAS) to the biologically active dehydroepiandrosterone (DHEA) was investigated in mixed synovial cells. The effects of DHEA on PlGF-1 expression were studied by intracellular fluorescence-activated cell sorting analysis. RESULTS: PlGF-1-positive cells were detected in the lining and sublining areas in patients with RA and patients with OA, and cellular density was similar. Double staining revealed that PlGF-1-positive cells were macrophages. In RA and OA, the density of PlGF-1-positive cells correlated positively with the density of macrophages and the density of type IV collagen-positive vessels. The supernatant concentration of (3) H-DHEA after conversion from (3) H-DHEAS and the density of aromatase-positive cells were positively correlated with the density of PlGF-1-positive cells only in OA. Low DHEA concentrations (≤10(-9) M) had stimulatory effects on PlGF-1 when compared to serum concentrations (10(-8) M to 10(-7) M) in the monocytic cell line THP-1 and in primary mixed synovial cells. CONCLUSION: PlGF-1 functions similarly in inflamed synovium and in the placenta. It is related to vessel formation and, in OA patients, to androgen/estrogen conversion. Evolutionarily conserved functions of PlGF-1 for placental phenomena are obviously also present in synovial inflammation.

Tubulin inhibitor AEZS 112 inhibits the growth of experimental human ovarian and endometrial cancers irrespective of caspase inhibition.

AEZS 112 is an orally active small molecule anticancer drug which inhibits the polymerization of tubulin at low micromolar concentrations. The current study investigates the anti-tumor effect and the mechanism of action of AEZS 112 in in vitro models of human ovarian and endometrial cancers. Four human ovarian and 2 endometrial cancer cell lines were incubated with increasing concentrations of AEZS 112 with and without multi-caspase inhibitor zVAD-FMK for 72 hours. Cytotoxic effects of AEZS 112 were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. AEZS 112 displayed anti-tumor activity in all six cell lines. The EC50 determined after 72-h incubation for Ishikawa and HEC 1A was 0.0312 and 0.125 microm, respectively. The EC50 was 5 microm for SKOV 3 cells, 1 microm for 0.5 microm for OAW 42 cells, 0.125 microm for OvW 1 cells and 0.0312 microm for PA 1 cells. Cytotoxic effects of AEZS 112 could not be abrogated by caspase inhibition with pan-caspase inhibitor zVAD-fmk. Annexin V/propidium iodide-double staining after treatment with AEZS 112 was indicative of necrosis-like cell death. AEZS 112 dose-dependently increased non-vital hypodiploid cells and the cytotoxic effect was least pronounced in G2 phase of the cell cycle, indicating cell death during mitosis, as determined by FACS analysis. The orally active small molecule tubulin inhibitor AEZS 112 showed anti-tumor activity in human ovarian and endometrial cancer cell lines at low micromolar concentrations, which could not be abrogated by caspase inhibition and is therefore a good candidate for in vivo studies in these tumors.

Perifosine inhibits growth of human experimental endometrial cancers by blockade of AKT phosphorylation.

OBJECTIVE: Perifosine is an orally active alkylphospholipid analog, which has shown anti-tumor activity in a variety of cancers by inhibition of AKT phosphorylation. The objective of the current study was to evaluate its efficacy in in vitro models of human endometrial cancer. STUDY DESIGN: The effect of 10microM and 40microM perifosine on AKT phophorylation in human endometrial cancer cell lines Ishikawa and HEC 1A was determined by Western blotting. To screen for a putative anti-tumor effect, HEC 1A and Ishikawa cells were incubated with increasing concentrations of perifosine for 24h, 48h and 72h and the number of viable cells was determined by crystal violet staining. Also the effect of a combined treatment with cisplatin and perifosine was investigated in Ishikawa cells. Flow cytometric analysis of DNA content was used to determine the effect of perifosine on the cell cycle distribution of HEC 1A and Ishikawa cells and to assess potential toxic side effects of perifosine on peripheral blood lymphocytes (PBL). RESULTS: AKT phosphorylation was dose-dependently inhibited by perifosine. Concomitantly, perifosine displayed anti-tumor activity in both cell lines at concentrations that showed no effect on peripheral blood lymphocytes. Growth inhibitory effects became more pronounced with increasing treatment time. While IC 50 values at 24h were >40microM, IC 50 values after 48h were approximately 7microM in Ishikawa and 25microM in HEC 1A cells. After 72h, the IC 50 was below 1.25microM for Ishikawa and about 6microM for HEC 1A cells. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in human Ishikawa endometrial cancer cells. Of note, the anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle. CONCLUSIONS: The small molecule AKT inhibitor perifosine showed substantial anti-tumor activity in human endometrial cancer cell lines. Since these effects were increased with cisplatin, perifosine seems to be a good candidate for treatment combinations with classical cytostatic compounds. Thus, perifosine should be further evaluated in clinical studies in endometrial cancer.

Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by down-regulating NKG2D.

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.

Inflammation and sex hormone metabolism.

The incidence of autoimmune diseases is higher in females than in males. In both sexes, adrenal hormones, that is, glucocorticoids, dehydroepiandrosterone (DHEA), and androgens, are inadequately low in patients when compared to healthy controls. Hormonally active androgens are anti-inflammatory, whereas estrogens are pro-inflammatory. Therefore, the mechanisms responsible for the alterations of steroid profiles in inflammation are of major interest. The local metabolism of androgens and estrogens may determine whether a given steroid profile found in a subject's blood results in suppression or promotion of inflammation. The steroid metabolism in mixed synovial cells, fibroblasts, macrophages, and monocytes was assessed. Major focus was on cells from patients with rheumatoid arthritis (RA), while cells from patients with osteoarthritis served as controls. Enzymes directly or indirectly involved in local sex steroid metabolism in RA are: DHEA-sulfatase, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and aromatase (CYP19), which are required for the synthesis of sex steroids from precursors, 5alpha-reductase and 16alpha-hydroxylase, which can be involved either in the generation of more active steroids or in the pathways leading to depletion of active hormones, and 3alpha-reductase and 7alpha-hydroxylase (CYP7B), which unidirectionally are involved in the depletion of active hormones. Androgens inhibit aromatization in synovial cells when their concentration is sufficiently high. As large amounts of estrogens are formed in synovial tissue, there may be a relative lack of androgens. Production of 5alpha-reduced androgens should increase the local anti-inflammatory activity; however, it also opens a pathway for the inactivation of androgens. The data discussed here suggest that therapy of RA patients may benefit from the use of nonaromatizable androgens and/or the use of aromatase inhibitors.

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes--androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5alpha-reduced androgens.

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16alphaOH-DHEA, 7alphaOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16alphaOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16alpha-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5alpha-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5alpha-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16alphaOH-testosterone. 5alpha-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5alpha-dihydro-ASD, 5alpha-dihydrotestosterone, and negligible quantities of E1 and E2. 5alpha-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5alpha-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16alpha-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.

Ablation of the sympathetic nervous system decreases gram-negative and increases gram-positive bacterial dissemination: key roles for tumor necrosis factor/phagocytes and interleukin-4/lymphocytes.

The sympathetic nervous system is intensely activated during bacteremia, but its immediate influence on the bacterial tissue burden remains unclear. We demonstrate that prior ablation of the sympathetic nervous system decreases this dissemination of Pseudomonas aeruginosa or Escherichia coli through a mechanism of increased secretion of peritoneal tumor necrosis factor, improved phagocytic response of peritoneal cells, and increased influx of monocytes into the peritoneal cavity. When gram-positive Staphylococcus aureus strains were used, sympathectomy increased the bacterial tissue burden, which was caused by a reduction in corticosterone tonus, and decreased both interleukin-4 secretion from peritoneal cells and the influx of lymphocytes into the peritoneal cavity. In both models, the peritoneal wall was the critical border for systemic infection. These results show the dual role of the sympathetic nervous system in sepsis. It can be favorable or unfavorable, depending on the innate immune effector mechanisms necessary to overcome infection.

Sex hormone concentrations in patients with rheumatoid arthritis are not normalized during 12 weeks of anti-tumor necrosis factor therapy.

OBJECTIVE: Androgens such as dehydroepiandrosterone sulfate (DHEAS) and testosterone are markedly lower in postmenopausal women with rheumatoid arthritis (RA) than in controls. In contrast, compared to controls, serum levels of estrogens are normal or elevated in women with RA. Since tumor necrosis factor (TNF) alters production of these hormones, we investigated changes of these hormones during anti-TNF antibody (anti-TNF) therapy with adalimumab in longstanding RA. METHODS: In this longitudinal anti-TNF therapy study in 13 patients with long-standing RA without prior prednisolone (7 infusions of anti-TNF: Week 0, 2, 4, 6, 8, 10, and 12), we measured serum concentrations of interleukin 6 (IL-6), androstenedione, DHEA, DHEAS, free testosterone, estrone, and 17ss-estradiol. Levels of these hormones in patients were compared to serum levels of 31 age and sex matched healthy controls. RESULTS: Upon treatment with anti-TNF, there was an impressive decrease of clinical markers of inflammation, erythrocyte sedimentation rate, and serum levels of IL-6. Serum levels of DHEAS and free testosterone were markedly lower at baseline in patients compared to controls, but this did not change during anti-TNF therapy. Serum levels of DHEA and 17ss-estradiol were significantly elevated in patients compared to controls, but similarly, anti-TNF therapy did not change initially increased levels. Molar ratios of hormones, which reflect hormone shifts via converting enzymes, showed typical alterations at baseline, but did not change markedly during anti-TNF therapy. CONCLUSION: Longterm therapy with anti-TNF did not change altered serum levels of typical sex hormones in patients with RA, although baseline values were largely different. In patients with RA, this indicates that alterations of sex hormones and altered activity of respective converting enzymes are imprinted for a long-lasting period over at least 12 weeks.

Tumor necrosis factor inhibits conversion of dehydroepiandrosterone sulfate (DHEAS) to DHEA in rheumatoid arthritis synovial cells: a prerequisite for local androgen deficiency.

OBJECTIVE: Use of anti-tumor necrosis factor (anti-TNF) antibody therapy in rheumatoid arthritis (RA) has expanded our understanding of possible mechanisms by which this treatment reduces inflammation. Beyond its effects on local immune responses, anti-TNF treatment may also modulate the local hormone supply. Because androgens are thought to inhibit immune responses, their presence in inflamed tissue is an additional important antiinflammatory factor. METHODS: We investigated conversion of the ubiquitous dehydroepiandrosterone sulfate (DHEAS), the biologically inactive precursor of DHEA, to the androgen DHEA in mixed synovial cells from patients with RA and patients with osteoarthritis (OA), making use of thin-layer chromatography and phosphorimaging. Using immunohistochemical analysis, we detected the key enzyme, steroid sulfatase. RESULTS: DHEAS-to-DHEA conversion in synovial cells from patients with RA was significantly lower than that in synovial cells from patients with OA (mean +/- SEM 3.3 +/- 0.5% versus 6.0 +/- 0.9% of applied (3)H-DHEAS per 10(6) synovial cells; P = 0.042). In RA, but not in OA, the level of converted (3)H-DHEA was inversely correlated with the density of synovial macrophages (for RA, R(rank) = -0.725, P = 0.005; for OA, R(rank) = 0.069, P not significant [NS]) and T cells (for RA, R(rank) = -0.621, P = 0.024; for OA, R(rank) = 0.247, P NS). Double immunohistochemistry analysis revealed that steroid sulfatase was located mainly in synovial macrophages but was also observed in fibroblasts. Neutralization of TNF largely up-regulated the conversion of DHEAS to DHEA in RA, but not in OA. A similar neutralizing effect was observed with polyclonal human immunoglobulins; this effect is most probably mediated via TNF neutralization at low TNF concentrations. CONCLUSION: These data indicate that TNF inhibits the conversion of DHEAS to DHEA in RA synovial cells. Because androgens are antiinflammatory mediators, TNF-induced inhibition of the local androgen supply is a supplementary proinflammatory factor. Consequently, anti-TNF strategies may also exert their positive effects by increasing tissue androgens.

Reduced capacity for the reactivation of glucocorticoids in rheumatoid arthritis synovial cells: possible role of the sympathetic nervous system?

OBJECTIVE: Cortisol, the biologically active glucocorticoid, is a major endogenous antiinflammatory factor in rheumatoid arthritis (RA). The aim of this study was to examine the local conversion of cortisol to biologically inactive cortisone and vice versa (the cortisol-cortisone shuttle) in RA and osteoarthritis (OA) patients. METHODS: Thin-layer chromatography and phosphorimaging were used to examine the cortisol-cortisone shuttle in mixed synovial cells. Double immunohistochemistry was used to assess the key enzymes 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) and 11beta-HSD2 and their possible cellular locations. RESULTS: Double immunohistochemistry demonstrated 11beta-HSD1/2+ macrophages in the sublining area. The ratio of 11beta-HSD2+ cells to 11beta-HSD1+ cells was significantly higher in RA than in OA patients. Cortisol was converted to inactive cortisone in mixed synovial cells from RA and OA patients, which was largely inhibited by carbenoxolone (11beta-HSD1 and 11beta-HSD2 inhibitor). Using metyrapone to inhibit the 11beta-HSD1 reducing reaction (cortisone --> cortisol), we demonstrated that the capacity for reactivation of cortisone to cortisol was significantly higher in OA than in RA patients. Although the capacity for the cortisone-cortisol shuttle was higher in synovial cells from less-inflamed OA tissue compared with inflamed RA tissue, it was obvious that synovial inflammation in RA, but not OA, was related positively to the reactivation of cortisone. This indicates that in RA, a cause other than typical inflammatory factors inhibits the reactivation of cortisone. Since isoproterenol and adenosine inhibited the cortisol-cortisone shuttle, the loss of sympathetic nerve fibers (loss of beta-adrenergic agonist and adenosine) may be the missing link that accounts for the increased cortisol-cortisone shuttle in RA. CONCLUSION: This study demonstrates a reduced capacity for local reactivation of cortisone in RA synovial cells. Since synthetic glucocorticoids also use this reactivation shuttle, the results also apply to therapeutic glucocorticoids. This defective reactivation of cortisone may be an important unrecognized pathophysiologic factor in RA.

Increased prevalence of semaphorin 3C, a repellent of sympathetic nerve fibers, in the synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: The presence of selective sympathetic nerve repellents, i.e., semaphorins, may be responsible for the observed reduction of sympathetic innervation in the synovial tissue of patients with rheumatoid arthritis (RA). This study was undertaken to investigate the presence of different semaphorins in synovial tissue of patients with RA, patients with osteoarthritis (OA), and control subjects without inflammation. METHODS: In situ hybridizations with digoxigenin-labeled RNA probes directed against different semaphorins were performed. The presence of semaphorin 3C (S3C) in the synovial tissue of 10 RA, 10 OA, and 5 control subjects was investigated using a polyclonal antiserum directed against S3C. RESULTS: All in situ hybridizations revealed the presence of S3C messenger RNA, but no other investigated semaphorin (i.e., against primary afferent sensory nerve fibers), in the synovial tissue of RA and OA patients. Immunohistologic double staining demonstrated that macrophages and fibroblasts were positive for S3C protein. Quantitative analysis of S3C protein staining showed an increased density of S3C-positive cells in the synovial tissue of RA patients (mean +/- SEM 339 +/- 65 cells/mm(2)) in comparison with OA patients (168 +/- 27/mm(2); P = 0.031 versus RA) and controls (126 +/- 26/mm(2); P = 0.027 versus RA). Studies of the relationship between sympathetic nerve fiber density and S3C-positive cell density in the tissue of all patients showed that RA patients generally had lower densities of sympathetic nerve fibers and higher densities of S3C-positive cells than OA patients and control subjects. CONCLUSION: These findings suggest that S3C from macrophages and fibroblasts, which is selectively directed against sympathetic nerve fibers, could be one element responsible for reduced sympathetic innervation in RA tissue. The inability of sympathetic nerve fibers to reinnervate synovial tissue could contribute to the chronic nature of RA.

Patients with rheumatoid arthritis and systemic lupus erythematosus have increased renal excretion of mitogenic estrogens in relation to endogenous antiestrogens.

OBJECTIVE: In patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), 17beta-estradiol was thought to play a dual pro- and antiinflammatory role depending on its concentration or probably conversion to downstream mitogenic 16 alpha-hydroxyestrone or naturally occurring antiestrogens such as 2-hydroxyestrone. We compared renal excretion of these 2 types of estrogens in healthy subjects and patients with RA and SLE. METHODS: In a prospective study with 30 patients with RA, 32 with SLE, and 54 healthy subjects, we measured urinary levels of 16 alpha-hydroxyestrone and 2-hydroxyestrogens by enzyme immunoassay. We studied renal excretion to estimate the time-integral of hormone production. RESULTS: Urinary concentration and total urinary loss of 2-hydroxyestrogens was 10 times higher in healthy subjects compared to patients with either SLE or RA irrespective of prior prednisolone treatment or sex. The urinary concentration and loss of 16 alpha-hydroxyestrone did not differ between healthy subjects and patients with RA/SLE. The ratio of urinary 16 alpha-hydroxyestrone/2-hydroxyestrogens was more than 20 times higher in RA and SLE than healthy subjects irrespective of prior glucocorticoid treatment or sex. CONCLUSION: This study in RA and SLE patients clearly demonstrates a large shift to mitogenic estrogens in relation to endogenous antiestrogens. Both steroids are converted from the precursor 17beta-estradiol and estrone. In patients with RA and SLE, the magnitude of conversion to the mitogenic 16 alpha-hydroxyestrone is greatly upregulated, which likely contributes to maintenance of the proliferative state in these diseases.