Synthetic triterpenoids, CDDO-Imidazolide and CDDO-Ethyl amide, induce chondrogenesis.

Novel methods for inducing chondrogenesis are critical for cartilage tissue engineering and regeneration. Here we show that the synthetic oleanane triterpenoids, CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide (CDDO-EA), at concentrations as low as 200 nM, induce chondrogenesis in organ cultures of newborn mouse calvaria. The cartilage phenotype was measured histologically with metachromatic toluidine blue staining for proteoglycans and by immunohistochemical staining for type II collagen. Furthermore, real-time polymerase chain reaction (PCR) analysis using mRNA from calvaria after 7-day treatment with CDDO-Im and CDDO-EA showed up-regulation of the chondrocyte markers SOX9 and type II collagen (alpha1). In addition, TGF-ß; BMPs 2 and 4; Smads 3, 4, 6, and 7; and TIMPs-1 and -2 were increased. In contrast, MMP-9 was strongly down-regulated. Treatment of human bone marrow-derived mesenchymal stem cells with CDDO-Im and CDDO-EA (100 nM) induced expression of SOX9, collagen IIα1, and aggrecan, as well as BMP-2 and phospho-Smad5, confirming that the above triterpenoids induce chondrogenic differentiation. This is the first report of the use of these drugs for induction of chondrogenesis.

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

[Ser77]transforming growth factor-beta 1. Selective biological activity and receptor binding in mink lung epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) is a homodimeric protein stabilized by a single disulfide bridge between Cys77 on the respective monomers and two paired complementary hydrophobic interfaces between the two subunits. A TGF-beta 1 mutant with Cys77 replaced by serine has been expressed in stably transfected Chinese hamster ovary cells and purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirms that the sole interchain disulfide bond in TGF-beta 1 has been eliminated. It is 20% as potent as native TGF-beta 1 in the induction of plasminogen activator inhibitor-1 promoter expression in mink lung epithelial cells (Mv1Lu), although it is less than 1% as potent as native TGF-beta 1 in inhibition of growth in the same cell line. The mutant acts as a full agonist in both bioassays. [Ser77]TGF-beta 1 binds to soluble type II receptors and competes with native TGF-beta 1 in sandwich-enzyme-linked immunosorbent assays; however, in Mv1Lu cells, the mutant shows preferential cross-linking to type I rather than type II receptors. [Ser77]TGF-beta 1 is a useful tool for understanding the different ligand-receptor complexes and numerous biological activities of this multifunctional cytokine.

Prevention of breast cancer in the rat with 9-cis-retinoic acid as a single agent and in combination with tamoxifen.

We show that 9-cis-retinoic acid (9cRA) is a potent inhibitor of mammary carcinogenesis induced by N-nitroso-N-methylurea in Sprague-Dawley rats. Rats were first treated with a single dose of N-nitroso-N-methylurea (50 mg/kg body weight) and then fed non-toxic levels of 9cRA (120 or 60 mg/kg of diet). 9cRA was highly effective in reducing tumor incidence, average number of tumors per rat, and average tumor burden, as well as extending tumor latency. The combination of 9cRA with low levels of tamoxifen (TAM; fed at either 1.0 or 0.5 mg/kg of diet) was particularly effective; addition of 9cRA to a TAM regimen doubled the number of animals that were tumor-free at autopsy and significantly diminished tumor number and tumor burden. For suppression of carcinogenesis in vivo, 9cRA was much more potent than all-trans-retinoic acid, both as a single agent or in combination with TAM, although both retinoids had equivalent inhibitory effects on DNA synthesis in cultured human breast cancer cell lines. Both 9cRA and all-trans-retinoic acid induce the expression of the adhesion molecule, E-cadherin, in the SK-BR-3 cell line. We suggest that clinical evaluation of the combination of 9cRA and TAM, either for chemoprevention or for adjuvant therapy, should be considered.

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization.

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.

Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.

Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with 20% serum or in basal media supplemented with platelet-derived growth factor (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal growth factor (EGF), interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) did not support growth, although EGF enhanced PDGF-dependent growth. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony growth. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic fibroblast-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of fibroblast-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine growth factors, such as PDGF and TGF-beta.

Effects of retinoid deficiency on the development of the heart and vascular system of the quail embryo.

The regulatory role of retinoids in growth and differentiation has been examined in vitro and in vivo by light and scanning electron microscopy using retinoid-deficient and control quail embryos between the 5-15 somite stage, as well as 2- and 2.5-day-old embryos. Fertile, retinoid-deficient eggs were obtained from flocks of quail maintained on a retinoid- and carotenoid-deficient diet, supplemented only with small amounts of retinoic acid methyl ester as described by Thompson et al. 1969. As described previously, retinoid deprivation during embryonal development causes abnormalities in organs of epithelial and mesenchymal origin, most dramatically preventing the formation of the extraembryonal circulatory system in the avian embryo. Our in vivo studies show that the basis for the latter defect is the failure of the primitive heart tubes to open at their posterior end, thus preventing the formation of omphalomesenteric veins normally connecting the embryonal with the extraembryonal circulatory system. Early manifestation of the retinoid-deficient defect may result also in formation of a cardia bifida, late manifestation in development of a single dilated ventricle. In contrast, the extraembryonal vascular system of blood islands is well developed. Heart function as shown by the rate of heart beat is reduced in deficient embryos. Our in vitro studies demonstrate similar defects in the development of the circulatory system by culture of normal 24-h embryos on retinoid-deficient agar medium; conversely, normal development is observed upon culture of retinoid-deficient embryos on retinoid-containing agar medium.

N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder cancer in C57BL/6 X DBA/2 F1 mice as a useful model for study of chemoprevention of cancer with retinoids.

Highly invasive carcinomas of the urinary bladder were induced in male C57BL/6 X DBA/2 F1 (hereafter called B6D2F1) mice by gastric intubation of N-butyl-(4-hydroxybutyl)nitrosamine (OH-BBN) using a quantitative dosing schedule. Animals received either 5 or 10 mg OH-BBN per intubation, two times each week, for 9 weeks for a total dose of either 90 or 180 mg, and they were killed 6 months after the first carcinogen intubation. Seven days after the final intubation of OH-BBN, animals were fed either a placebo diet or diet supplemented with either 150 or 200 mg 13-cis-retinoic acid per kg of diet. A 41 and 43% incidence of urinary bladder cancer was observed in mice given the low and high dose of carcinogen, respectively, and fed a placebo diet. Sixty-seven % of the carcinomas induced in these animals invaded either into or through the urinary bladder wall. Varying degrees of transitional and either squamous or glandular or both squamous and glandular differentiation were observed in the carcinomas. Feeding of diet supplemented with 13-cis-retinoic acid reduced cancer incidence; the degree of reduction was proportional to the dose of retinoid administered. The highly invasive nature of the carcinomas induced by quantitative administration of OH-BBN in B6D2F1, mice provides a useful animal model of the highly invasive variant of human transitional cell urinary bladder cancer in which to study chemoprevention by retinoids as well as other compounds.

Inhibition of urinary bladder cancer by N-(ethyl)-all-trans-retinamide and N-(2-hydroxyethyl)-all-trans-retinamide in rats and mice.

The chemopreventive activity of two synthetic retinamides of relatively low toxicity against N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder cancer was studied in F344 rats and C57BL/6 X DBA/2 F1 mice. Female and male rats were given a total dose of either 1800 or 3200 mg OH-BBN over a period of 6 or 8 weeks, respectively. Male mice were given a total dose of either 90 or 180 mg OH-BBN over a period of 9 weeks. Seven days after the final intubation of a period of 9 weeks. Seven days after the final intubation of OH-BBN, animals were fed either a placebo diet or a diet supplemented with the following retinoids: for rats, 0.8 mmol 13-cis-retinoic acid, 2 mmol N-(ethyl)-all-trans-retinamide, or 2 mmol N-(2-hydroxyethyl)-all-trans-retinamide per kg diet; and for mice, either 0.5 or 1.0 mmol of N-(ethyl)-all-trans-retinamide or N-(2-hydroxyethyl)-all-trans-retinamide per kg diet. Animals were killed 6 months after the initial gastric intubation. In comparison to male and female rats fed placebo diets, all three retinoids reduced the incidence, number, and severity of the low-grade papillary transitional cell carcinomas of the urinary bladder. Similarly, treatment of mice with either of the two retinamides reduced the incidence of highly invasive urinary bladder carcinomas. The chemopreventive effect of the less toxic retinamides was equal to or greater than that of 13-cis-retinoic acid.

Inhibitory effect of 13-cis-retinoic acid on urinary bladder carcinogenesis induced in C57BL/6 mice by N-butyl-N-(4-hydroxybutyl)-nitrosamine.

The effect of 13-cis-retinoic acid on the induction of urinary bladder carcinoma by N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) was studied in male C57BL/6 mice. Animals received a total dose of either 90 or 140 mg of OH-BBN via gastric intubations of 7.5 or 10.0 mg of OH-BBN 2 times each week for 6 or 7 weeks, respectively. Seven days after the last OH-BBN intubation, animals were fed laboratory chow diet supplemented with either 200 mg of 13-cis-retinoic acid per kg or its placebo. Animals were killed at 6 months after the first carcinogen intubation. Highly invasive squamous and transitional cell carcinomas of the urothelium were found at autopsy. In the majority of these carcinomas, invasion of the bladder muscle wall by tumor cells had occurred. At the two dose levels of OH-BBN, feeding of 13-cis-retinoic acid reduced the incidence of both carcinomas and noninvasive papillomas, as well as the extent of neoplastic development in the urinary bladder. In mice receiving the lower dose of OH-BBN, the feeding of 13-cis-retinoic acid prevented the appearance of both squamous and transitional cell carcinomas with a reduction in incidence from 33 to 0% (p less than 0.01). The results of this study indicate that 13-cis-retinoic acid reduced not only the severity of highly invasive urinary bladder carcinomas but also the incidence of such cancers.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis by retinyl acetate.

The administration of 2.5 mg retinyl acetate daily in the diet to female Sprague-Dawley rats beginning 7 days after the intragastric instillation of either 2.5, 5, or 15 mg 7,12-dimethylbenz(a)anthracene (CMBA) resulted in a reduction in the incidence of benign mammary tumors of 37, 30, and 31%, respectively. An equally significant reduction in the number of tumors was also evident. Although no difference was noted in the percentage incidence of mammary adenocarcinomas between the placebo and 2.5 mg retinyl acetate-treated groups at the 2.5-mg DMBA level, the percentage incidence was reduced by 52 and 39% in these groups at the 5- and 15-mg DMBA dose. Furthermore, the number of adenocarcinomas was also significantly reduced. Although both the percentage incidence and number of tumors were reduced by treatment with 1 mg retinyl acetate, these differences were not statistically significant. Liver histology and liver function tests of rats of the retinyl acetate groups did not differ from that of the control group. Similarly, the estrus cycle of treated animals did not differ from that of control rats. These data indicate that relatively large doses of retinyl acetate significantly inhibit the development of DMBA-induced mammary adenocarcinomas and benign tumors. Furthermore, the suppression of mammary tumorigenesis is apparently not the result of an alteration in either the metabolism of DMBA or estrogen nor to an inhibition of tumor growth resulting from retinyl acetate toxicity. The inhibitory effect of retinyl acetate may be related to the effect of retinoids on epithelial cell differentiation and/or reversal of carcinogen-induced anaplasia.