Mice null for NEDD9 (HEF1α) display extensive hippocampal dendritic spine loss and cognitive impairment.

NEDD9 (neural precursor cell expressed, developmentally down-regulated 9) is a member of the CAS (Crk-associated substrate) family of scaffolding proteins that regulate cell adhesion and migration. A Nedd9 knock-out/lacZ knock-in mouse (Nedd9(-/)(-)) was developed in order to study Nedd9 expression and function in the nervous system. Herein we show that NEDD9 is expressed in the adult brain and is prominently expressed in the hippocampus. Behavioral testing uncovered functional deficits in Nedd9(-)(/)(-) mice. In the Morris water maze test, Nedd9(-)(/)(-) mice showed deficits in both the ability to learn the task as well as in their ability to recall the platform location. There was no change in the gross morphology of the hippocampus, and stereological analysis of BrdU-labeled newly formed hippocampal cells suggested that this defect is not secondary to altered neurogenesis. However, analysis of the hippocampus revealed extensive loss of dendritic spine density in both the dentate gyrus (DG) and CA1 regions. Spine loss occurred equally across all branch orders and regions of the dendrite. Analysis of spine density in Nedd9(-)(/)(-) mice at 1.5, 6 and 10 months revealed an age-dependent spine loss. This work shows that NEDD9 is required for the maintenance of dendritic spines in the hippocampus, and suggests it could play a role in learning and memory.

The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation.

Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.

atRA Regulation of NEDD9, a gene involved in neurite outgrowth and cell adhesion.

We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from -1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from -475 to -445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.

Vascular tube formation on matrix metalloproteinase-1-damaged collagen.

Connective tissue damage and angiogenesis are both important features of tumour growth and invasion. Here, we show that endothelial cells maintained on a three-dimensional lattice of intact polymerised collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human basal cell tumours of the skin (containing a high level of active matrix metalloproteinase-1 (MMP-1)), degradation of the collagen matrix occurred. The major degradation products were the $3 over 4$- and $1 over 4$-sized fragments known to result from the action of MMP-1 on type I collagen. When endothelial cells were maintained on the partially degraded collagen, the cells organised into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase-1 (TIMP-1) or neutralising antibody to MMP-1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organisation into the vascular network. Purified (activated) MMP-1 duplicated the effects of skin organ culture fluid, but other enzymes including MMP-9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and also failed to promote vascular tube formation. Together, these studies suggest that damage to the collagenous matrix is itself an important inducer of new vessel formation.

2MD, a new anabolic agent for osteoporosis treatment.

INTRODUCTION: 2-Methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD) is a new analog of 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that has unique properties (distinct from 1alpha,25-dihydroxyvitamin D3) in stimulating osteoblasts to form bone in culture. This analog has now been extensively tested in aged ovariectomized female rats maintained on a diet adequate in calcium and phosphorus. METHODS: Retired female rats obtained from Sprague-Dawley were ovariectomized, and were either dosed with vehicle or 2MD at 5-7 ng/kg body weight each day. RESULTS: A marked increase in total bone mass resulted during the 28-week study. This increase in bone mass resulted from an increase in both cortical and trabecular bone, with increases to the order of 25% in the cancellous bone. Histomorphometry revealed that 2MD increased bone mass primarily by increasing bone formation. It also revealed little or no effect on bone resorption. The resulting bone is of high quality revealed by histology and biomechanical testing. CONCLUSION: Throughout the study, serum calcium remained within the normal range and thus 2MD shows great promise for the treatment of bone diseases characterized by bone loss, including osteoporosis.

A mammalian homolog of unc-53 is regulated by all-trans retinoic acid in neuroblastoma cells and embryos.

The vitamin A metabolite, all-trans retinoic acid (atRA), plays an important role in neuronal development, including neurite outgrowth. However, the genes that lie downstream of atRA and its receptors in neuronal cells are largely unknown. By using the human neuroblastoma cell line, SH-SY5Y, we have identified an atRA-responsive gene (RAINB1: retinoic acid inducible in neuroblastoma cells) that is induced within 4 h after exposure of SH-SY5Y cells to atRA. RAINB1 mRNA is highly expressed in the nervous system (10.5- to 11-kb transcript) in both developing embryos and adults. Its expression is perturbed in developing rat embryos exposed to excess or insufficient atRA. RAINB1 is present on chromosome 11 and is spread over 38 exons, resulting in a putative ORF of 2,429 amino acids. The RAINB1 protein shows high similarity to a gene in Caenorhabditis elegans, unc-53, that is required for axonal elongation of mechanosensory neurons, suggesting that these proteins are orthologs. Thus, RAINB1 may represent a critical downstream gene in atRA-mediated neurite outgrowth.

Induction of matrix metalloproteinase-1 (MMP-1) during epidermal invasion of the stroma in human skin organ culture: keratinocyte stimulation of fibroblast MMP-1 production.

Organ cultures of human skin were incubated for 8 days under growth factor-free conditions or exposed to 10 ng ml(-1) of human recombinant epidermal growth factor (EGF) during the incubation period. Normal histological features were preserved in the absence of growth factor, while epithelial cells underwent a proliferative response and invaded the underlying stroma in the presence of exogenous EGF. The same concentrations of EGF that induced stromal invasion also resulted in up-regulation of matrix metalloproteinase-9 (MMP-9; 92-kD gelatinase B) in organ culture and keratinocyte monolayer culture, and expression of MMP-1 (interstitial collagenase) in organ culture and fibroblast monolayer culture. When skin organ cultures were exposed to a potent, irreversible EGF-receptor tyrosine kinase (EGF-RTK) antagonist along with EGF, abnormal histological features were reversed, and MMP-9 production was suppressed. In contrast, EGF-RKT antagonism had only a modest inhibitory effect on MMP-1 production. Culture fluid from keratinocytes grown in monolayer culture stimulated fibroblast proliferation and MMP-1 elaboration. Treatment of fibroblasts with the same EGF-RTK antagonist inhibited keratinocyte-induced fibroblast proliferation but had only a modest inhibitory effect (approximately 20% inhibition) on MMP-1 production. In contrast, treatment of dermal fibroblasts with Interleukin-1 Receptor Antagonist had no effect on keratinocyte-induced fibroblast growth but strongly inhibited MMP-1 production (greater than 70% inhibition). These data indicate that stromal invasion by epithelial cells in EGF-treated skin is associated with events occurring in both the epidermis and dermis. The direct effect of the exogenous growth factor appears to be primarily on the epidermis. Dermal events reflect, at least in part, a response to factors elaborated in the epidermis.

Retinoic acid combined with neurotrophin-3 enhances the survival and neurite outgrowth of embryonic sympathetic neurons.

Both nerve growth factor (NGF) and neurotrophin-3 (NT-3) are necessary for the survival of embryonic sympathetic neurons in vivo. All-trans retinoic acid (atRA) has been shown to promote neurite outgrowth and long-term survival of chick embryonic sympathetic neurons cultured in the presence of NGF. The present study shows that atRA can also potentiate the survival and neurite outgrowth-promoting activities of NT-3. This was accomplished by enhancing the survival of existing neurons, as cell proliferation was unaffected by exposure to atRA. atRA also enhanced neurite outgrowth of the NT-3-treated cells; however, the neurites appeared thicker and less branched than cells treated with atRA in combination with NGF. Using a quantitative PCR assay, trkA and p75(NTR) mRNAs, but not trkC mRNA, were increased ( approximately 1.5- to 2-fold) after 72 and 48 hr of exposure of the cultures to atRA, respectively. The atRA-induced increase in trkA mRNA may play a role in the enhanced survival of neurons cultured in the presence of either NGF or NT-3, as both neurotrophins have been shown to signal through this receptor. The time course of these mRNA changes would indicate that atRA does not regulate the neurotrophin receptor mRNA directly, rather, intervening gene transcription is required. Thus, during development, atRA may play a role in fine-tuning embryonic responsiveness to both NT-3 and NGF.

An unhydrolyzable analogue of N-(4-hydroxyphenyl)retinamide. synthesis and preliminary biological studies.

The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic effects.

Matrix metalloproteinases (MMPs) in fresh human prostate tumour tissue and organ-cultured prostate tissue: levels of collagenolytic and gelatinolytic MMPs are low, variable and different in fresh tissue versus organ-cultured tissue.

Prostate tissue was obtained from 22 radical prostatectomies (performed for clinical management of prostate carcinoma) immediately after surgery. A small piece of tissue was fixed immediately in formalin and used for routine histology while a second piece was frozen in OCT and used for immuno-histochemistry. Another small piece was used for isolation of epithelial and stromal cells. The remainder of the tissue was cut into 2 x 2 mm pieces and incubated in organ culture for 8 days. In organ culture, non-malignant, basal epithelial cells underwent a proliferative response. This was accompanied by de-differentiation of glandular structures and by migration of epithelial cells across the surface of the tissue. Erosion of the basement membrane could also be seen in places, but was not widespread. Invasion of epithelial cells into the adjacent stroma was not evident. Production of matrix metalloproteinases (MMPs) with gelatinolytic activity or collagenolytic activity was assessed in organ culture and compared to expression patterns in fresh tissue. MMP-1 (interstitial collagenase) and MMP-9 (92-kDa gelatinase B) were undetectable or low in fresh tissue specimens. Both enzymes were detected in organ culture and both increased over time. Even after 6 days, however, there was only a low level of gelatin-hydrolytic activity and no measurable collagen-hydrolytic activity. In past studies we used organ cultures of normal skin and malignant skin tumours (basal cell carcinomas) to help elucidate the role of collagenolytic and gelatinolytic MMPs in epithelial cell invasion (Varani et al, 2000). Compared to MMP levels observed in skin, levels of these enzymes in prostate are low. The low level of collagenolytic and gelatinolytic MMPs in fresh prostate tissue and in organ-cultured prostate tissue may help explain why there is little tissue destruction in many primary prostate tumours and why the majority of such tumours remain confined to the prostate for extended periods.

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

Vitamin A controls epithelial/mesenchymal interactions through Ret expression.

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.

Heparin-binding epidermal-growth-factor-like growth factor activation of keratinocyte ErbB receptors Mediates epidermal hyperplasia, a prominent side-effect of retinoid therapy.

Sun-protected human skin was maintained in organ culture and treated with all-trans retinoic acid in the presence or absence of reversible or irreversible pharmacologic antagonists of c-erbB receptor tyrosine kinase activity. In the absence of these inhibitors, all-trans retinoic acid induced epidermal hyperplasia comparable to that induced in intact skin by all-trans retinol or all-trans retinoic acid itself. There was a strong correlation between inhibition of epidermal hyperplasia in organ culture and inhibition of epidermal-growth-factor-dependent keratinocyte growth in monolayer culture. In additional studies it was shown that all-trans retinoic acid could overcome the known inhibitory effects of calcium on expression of HB-EGF-like growth factor mRNA in organ-cultured skin. Further, it was shown that an antibody to HB-EGF-like growth factor inhibited retinoid-stimulated epidermal hyperplasia in organ culture and reduced proliferation in cultured keratinocytes. In contrast, the c-erbB receptor tyrosine kinase antagonists and the neutralizing HB-EGF-like growth factor antibody were ineffective in inhibiting all-trans-retinoic-acid-dependent survival and proliferation of human dermal fibroblasts. Taken together, these data indicate (i) that retinoid-induced epidermal hyperplasia in human skin proceeds through c-erbB, and (ii) that HB-EGF-like growth factor is one of the c-erbB ligands mediating this effect.

Chemotherapeutic evaluation of 4-hydroxybenzylretinone (4-HBR), a nonhydrolyzable C-linked analog of N-(4-hydroxyphenyl) retinamide (4-HPR) against mammary carcinogenesis.

The antitumor effects of N-(4-hydroxyphenyl)retinamide (4-HPR), and its stable C-linked analog, 4-hydroxybenzylretinone (4-HBR) on the regression of established 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors were compared. 4-HBR is a stable and nonhydroyzable derivative which cannot be converted in vivo to retinoic acid (RA). The results indicate that 4-HBR decreased mammary tumor volumes to the same extent as equimolar concentration (2 mmol/kg diet) of 4-HPR (-45% for 4-HBR vs. -42% for 4-HPR, p<0.01). Both 4-HPR and 4-HBR bind very poorly to nuclear retinoid receptors RARs and RXRs. The similarity of physicochemical properties of 4-HPR and 4-HBR as well as their equal antitumor potency suggests that 4-HPR like 4-HBR, is acting directly rather than through hydrolysis to free RA. Treatment with 4-HPR caused an almost 65% decrease in serum retinol levels. These results suggest that 4-HBR may have a significant chemotherapeutic advantage over 4-HPR, as the nonhydrolyzable analog may not cause night blindness which occurs as a significant side effect of 4-HPR usage.

Abnormal development of the sinuatrial venous valve and posterior hindbrain may contribute to late fetal resorption of vitamin A-deficient rat embryos.

BACKGROUND: Normal embryonic development and survival in utero is dependent on an adequate supply of vitamin A. Embryos from vitamin A-deficient (VAD) pregnant rats fed an inadequate amount of all-trans retinoic acid (atRA; 12 microg per g of diet or approximately 230 microg per rat per day) exhibit severe developmental abnormalities of the anterior cardinal vein and hindbrain by embryonic day (E) 12.5 and die shortly thereafter. METHODS: In the present study, we sought to determine whether supplementation of VAD-RA supported (12 microg per g of diet) pregnant rats with retinol (ROL) at the late-gastrula (presomite or rat E9.5) or early somite stages (E10.5), or provision of higher levels of atRA throughout this period could prevent abnormalities in the developing cardiovascular and nervous systems. RESULTS: A newly described defect in the sinuatrial venus valve along with enlarged anterior cardinal veins and nervous system abnormalities and the later death of embryos are prevented by supplementing pregnant animals with ROL on the morning of E9.5. If ROL supplementation is delayed by 1 day (E10.5), most embryos are abnormal and die by E18.5. Supplementation of VAD rats with atRA (250 microg per g of diet) between E8.5 and E10.5 also prevents the cardiovascular and nervous system abnormalities and a significant number of these embryos survive to parturition. Thus, high levels of atRA can obviate the need for ROL between E9.5 and E10.5. CONCLUSIONS: These results support an essential role for retinoid signaling between the late gastrula and early somite stages in the rat embryo for normal morphogenesis of the primitive heart tube and the posterior hindbrain. Further, these results suggest that embryonic death occurring at midgestation in the VAD rat may be linked to the abnormal development of one or both of these embryonic structures.

Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo.

The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5-like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal.

Vitamin A antagonizes the action of vitamin D in rats.

Interactions between vitamin A and vitamin D have been suggested for several decades but have not been established. In particular, vitamin A has been proposed to intensify the severity of the bone mineralization disease, rickets and inhibit the ability of vitamin D to cure this disease. To investigate this hypothesis, weanling Holtzman rats were fed a 1.2% calcium, 0.1% phosphorus diet and 15.5 ng ergocalciferol (vitamin D(2)) every 3 d for 21 d in the presence of increasing amounts of retinyl acetate (0 microg to 8621 microg/d). The increasing amounts of retinyl acetate produced a progressive and significant decrease in total bone ash (P < 0.001) and an increase in epiphyseal plate width (P < 0.001). The same experiment conducted with increasing amounts of vitamin D(2) (0 to 645 ng/d) indicated that the antagonism by retinyl acetate could be demonstrated at all vitamin D(2) dosages. To further investigate this antagonistic relationship, weanling Holtzman rats were fed a 0. 47% calcium, 0.3% phosphorus diet and 15.5 ng vitamin D(2) every 3 d for 33 d in the presence of increasing retinyl acetate (0 to 3448 microg/d). In the absence of retinyl acetate, these rats maintained a normal serum calcium level (2.34 mmol/L). Increasing retinyl acetate, however, eliminated the ability of vitamin D(2) to elevate the level of serum calcium (1.35 mmol/L). These results illustrated in vivo antagonism of vitamin D(2) action on intestine and bone by retinyl acetate.

Endothelial cell determinants of susceptibility to neutrophil-mediated killing.

Vascular endothelial cell injury plays an important role in the pathogenesis of inflammatory-mediated tissue injury. In the current study, we assessed injury in primary cultures of endothelial cells obtained from different sites within the same species, comparing rat dermal microvascular and rat lung microvascular endothelial cells. Dermal microvascular-derived endothelial cells were more sensitive to killing by PMA (phorbol myristate acetate)-activated human neutrophils than were endothelial cells derived from lung microvasculature. Lung endothelial cells stimulated with interferon-gamma plus lipopolysaccharide (IFNgamma + LPS) generated high levels of nitric oxide (*NO), while dermal endothelial cells stimulated with IFNgamma + LPS generated significantly lower levels of *NO. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of the *NO donor, S-nitroso-N-acetyl penicillamine (SNAP), endothelial cell killing by PMA-activated neutrophils was reduced. Lung endothelial cells stimulated with PMA generated less superoxide (02*-) than dermal endothelial cells. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of SNAP, O2*- release from endothelial cells was reduced. Endothelial cell-derived *NO appeared to play a significant role in attenuating the neutrophil-mediated killing. The differences in the ability of endothelial cells to generate *NO and 02*- underlies, at least in part, the differences in susceptibility of these cells to injury by activated neutrophils.

Chemopreventive activity of a C-glucuronide analog of N-(4-hydroxyphenyl) retinamide-O-glucuronide against mammary tumor development and growth.

The long term chemopreventive effects of the N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG), and its stable C-linked benzyl glucuronide analog, retinamidobenzyl glucuronide (4-HPRCG) on the growth and development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors were compared. The retinamidobenzyl glucuronide is stable toward acid hydrolysis and resists the actions of beta-glucuronidase. The results indicate that the C-linked glucuronide analog, 4-HPRCG has a greater chemopreventive potency than an equimolar concentration of 4-HPROG. Tumor latency was 15% longer in rats fed 2 mmol/kg diet of 4-HPRCG as compared to 4-HPROG. At 80 days post DMBA-intubation, tumor incidence was 57% and 27% in the 4-HPROG and 4-HPRCG treated rats, respectively. Tumor multiplicity was also markedly decreased in the 4-HPRCG treated rats. At 80 days post DMBA intubation the control rats had an average of 1.43 tumors/rat compared to 0.71 and 0.36 tumors/rat in the 4-HPROG and 4-HPRCG respectively. The higher potency and low toxicity of 4-HPRCG suggest that this stable analog may have an in vivo chemopreventive advantage over its analog, 4-HPROG. The results also demonstrated that these glucuronide analogs do not bind effectively in vitro either to the nuclear retinoid receptors or to the cellular retinoid binding proteins. Regardless of the mode of action of these retinoids, they are clearly effective chemopreventive agents.

Characteristics of nonmalignant and malignant human prostate in organ culture.

Prostate tissue was obtained from 52 radical prostatectomies immediately upon surgery. From each specimen, a small piece of tissue was fixed in 10% buffered formalin and used for histology, cytokeratin staining, staining with the antibodies to the proliferation-associated antigen (Ki-67), and histochemical evaluation of the epithelial-stromal basement membrane. A second piece was used for the isolation of epithelial cells and stromal cells in monolayer culture. The remainder of each specimen was cut into cubes (approximately 1 mm on a side) and incubated in organ culture for up to 20 days. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined as described above with zero-time tissue. These studies showed that normal epithelial and stromal elements survived in organ culture in the presence of a serum-free medium containing a mixture of growth factors (epidermal growth factor, insulin, pituitary extract, and dihydrotestosterone). In many of the tissues examined at 4 days, individual glands resembled those seen immediately after surgery, with a single layer of basal epithelial cells and a layer of secretory cells above. By Day 8, the secretory epithelium was lost in many places and basal cells proliferated to fill in the lumens of the glands. All of the nonmalignant glands were reactive with the anti-cytokeratin antibody (K903), and there was a large increase in the number of cells staining for Ki-67 as compared with zero-time tissue. Staining with the Periodic Acid Schiff (PAS) and PAS-methenamine silver (PASME) reagents revealed an intact basement membrane around virtually all of the epithelial structures. The basement membrane appeared to be thickened in some areas. In places where a gland was cut during the processing of the tissue, epithelial cells migrated out of the gland and covered the cut surface of the tissue piece. There was no detectable basement membrane separating the epithelium from the stroma at these sites. Whereas nonmalignant epithelial cells were preserved in the growth factor- and dihydrotestosterone-supplemented culture medium, most of the malignant cells rapidly lysed under the same conditions. However, when phorbol myristate acetate was included in the culture medium, many of the tumor cells remained viable. This was seen with the more well-differentiated tumors as well as with tumors that were highly anaplastic. All of the tumor cells were nonreactive with anti-cytokeratin antibody, and only a few cells stained for Ki-67. The basement membrane surrounding malignant cells was thin and, in places, appeared to be discontinuous. Where malignant glands were cut in the processing of the tissue, cells did not migrate out over the cut surface. In summary, this study identifies culture conditions for the successful maintenance of human prostate tissue for several days in organ culture. Histological/histochemical features that distinguish nonmalignant and malignant tissue are present in this model.