Robert J. Wordinger

Bone Morphogenetic Protein 4 inhibits TGF-beta2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation

The characteristic cupping of the optic nerve head (ONH) in glaucoma is associated with elevated TGF‐β2 and increased synthesis and deposition of extracellular matrix (ECM) proteins. In addition to TGF‐β2, the human ONH also expresses bone morphogenetic proteins (BMPs) and BMP receptors, which are members of the TGF‐β superfamily. We examined the potential effects of BMP4 and the BMP antagonist gremlin on TGF‐β2 induction of ECM proteins in ONH cells. BMP‐4 dose dependently inhibited TGF‐β2‐induced fibronectin (FN) and PAI‐1 expression in ONH astrocytes and lamina cribrosa (LC) cells and also reduced TGF‐β2 stimulation of collagen I, collagen VI, and elastin. Addition of gremlin blocked this BMP‐4 response, increasing cellular and secreted FN as well as PAI‐1 levels in both cell types. Gremlin was expressed in ONH tissues and ONH cells, and gremlin protein levels were significantly increased in the LC region of human glaucomatous ONH tissues. Interestingly, recombinant gremlin dose dependently increased ECM protein expression in cultured ONH astrocytes and LC cells. Gremlin stimulation of ECM required activation of TGF‐β receptor and R‐Smad3. TGF‐β2 increased gremlin mRNA expression and protein levels in ONH cells. Inhibition of either the type I TGF‐β receptor or Smad3 phosphorylation blocked TGF‐β2‐induced gremlin expression. In conclusion, BMP4 blocked the TGF‐β2 induction of ECM proteins in ONH cells. The BMP antagonist gremlin reversed this inhibition, allowing TGF‐β2 stimulation of ECM synthesis. Increased expression of gremlin in the glaucomatous ONH may further exacerbate TGF‐β2 effects on ONH ECM metabolism by inhibiting BMP‐4 antagonism of TGF‐β2 signaling. Modulation of the ECM via gremlin provides a novel therapeutic target for glaucoma.

Histology and ultrastructure of the adult mouse ovary following a single prenatal exposure to diethylstilbestrol

SummaryHistology, selective histochemistry and electron microscopy were used to examine the ovarian structure of offspring from mice administered DES (10 μg/kg in 0.1 cc of corn oil, subcutaneously) or corn oil alone on Day 15 of gestation. Offspring were sacrificed at 7 months of age. Ovarian changes in DES exposed offspring included the absence of distinguishable corpora lutea but the presence of follicles in various stages of growth and atresia. Large accumulations of pigmented cells and numerous enlarged, pale vacuolated interstitial cells were observed. Interstitial cells contained membrane bound vacuoles, lipid droplets and clumped pigmented material. A concentric pattern of membranes was often observed within the pigment. The results indicate that a single exposure to DES on Day 15 of gestation had a dramatic influence on ovarian morphology and function in 7 month old offspring. The ovarian morphology is consistent with tonic release of FSH and LH and failure in ovulation.

Transforming growth factor-β2 induces expression of biologically active bone morphogenetic protein-1 in human trabecular meshwork cells.

PURPOSE There are limited studies on the factors that regulate the processing of TGF-β2 and extracellular matrix (ECM) proteins into their mature form. Bone morphogenic protein 1 (BMP1) is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of our study was to determine whether cultured human trabecular meshwork (TM) cells express BMP1, BMP1 expression is regulated by TGF-β2, BMP1 is biologically active, and BMP1 regulates LOX activity. METHODS Primary human TM cells were isolated and subjected to quantitative PCR (qPCR) and Western immunoblotting (WB) for BMP1. BMP1 immunolocalization was performed in TM tissues. qPCR was used to determine BMP1 mRNA expression and WB results were used to determine BMP1 protein expression. BMP1 activity was measured in TM cells treated with TGF-β2 or with a combination of TGF-β2/UK383367. Lysyl oxidase (LOX) enzyme activity was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/β-aminoprorionitrile (BAPN). RESULTS Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-β2 increased mRNA expression compared to their controls (P<0.05). An ELISA showed TGF-β2-induced BMP1 secretion compared to their controls in all cell strains (P<0.05). Secreted BMP1 stimulated LOX enzymatic activity in TM cells. CONCLUSIONS BMP1 is expressed in the human TM. TGF-β2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms, resulting in TM stiffness and resistance to ECM degradation.

Histology of the adult mouse oviduct and endometrium following a single prenatal exposure to diethylstilbestrol

SummaryLight microscopy was used to examine the oviduct and endometrium of offspring from mice administered DES (10 μg/kg in 0.1 cc of corn oil, subcutaneously) or corn oil alone on Day 15 of gestation. Offspring were sacrificed at 5, 7 and 9 months of age. Oviduct changes in DES exposed offspring included numerous abnormal secretory cells which lined the mucosal folds of the isthmus. These cells contained a distinct granular cytoplasm which was eosinophilic and a nucleus displaced towards the apical surface. In addition both the ampulla and isthmus had mucosal folds which extended to the serosal surface and an accumulation of subepithelial fibrinoid material. Endometrial changes included squamous metaplasia of both the surface and glandular epithelial layer as well as extensive cystic glandular hyperplasia. In addition the endometrial connective tissue stroma exhibited fibrinoid accumulation. These changes may reflect an altered endocrine environment resulting from ovarian abnormalities during adulthood.

Uterine histamine content in mast-cell deficient (W/WV) mice during experimentally induced deciduoma formation

Abstract This study examines the changes in uterine histamine content in mast-cell normal and deficient mice during deciduoma formation. Surgical trauma (e.g., sutures) and intraluminal injection of oil produce deciduoma formation in ovariectomized mast-cell normal (+/+) and mast-cell deficient (W/WV) pseudopregnant mice that had previously received a single control ovary transplanted under the kidney capsule. Mast-cell normal (+/+) mice exhibit significant elevation of total uterine histamine content during both trauma and oil induced deciduoma formation. Mast-cell deficient (W/WV) mice under conditions of similar deciduoma formation did not show any change in total uterine histamine content. These results indicate that the change in histamine observed in mast-cell normal mice during deciduoma formation is most likely of mast cell origin. The lack of any measurable change in uterine histamine in mast-cell deficient mice indicates that an increase of non-mast cell histamine is not a prerequisite for deciduoma formation.

Sensitivity to diethylstilbestrol-(DES)-induced abortions is influenced by the whitten effect

Group-housed female mice, when introduced to a male, will undergo a synchronized estrous cycle with a characteristic pattern of mating where the majority of animals mate on the third day. This phenomenon is termed the Whitten effect. Exposure of pregnant mice to diethylstilbestrol (DES), a potent nonsteroidal estrogen, is capable of inducing premature parturition. The purpose of this study was to evaluate abortion induced by a single injection of DES on day 15 of gestation and compare the sensitivity depending on the day of mating. Initial observations confirmed that those animals that mated on day 3 were significantly more susceptible to DES-induced abortion than animals that mated on any other day. The possibility that this increase in sensitivity to abortion may be the result of differences in number or size of embryos at the time of DES exposure was examined. No significant difference was observed in the number of embryos at day 4 of gestation, at day 15 of gestation, or at birth. There was no difference in the weight of embryos at day 15 of gestation or at birth. A significant increase in the ratio of embryos between uterine horns was observed in the animals that mated on day 3 (ratio = 2.13) compared to animals mating on other days (ratio = 1.23 to 1.48). The cause of increased sensitivity to DES in animals that mated on day 3 is unclear. Our results may indicate that the induction of ovulation associated with the Whitten effect causes unequal embryo distribution leading to functional differences in sensitivity of the pregnancy to exogenous stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)