Growth In Organ Culture Research Articles

Effect of Platelet-Rich Plasma and other Key Factors on Hair Follicle Preservation.

Hair graft preservation is an important factor that influences graft survival in hair transplantation. To investigate the benefits of adding platelet-rich plasma (PRP), and the effect of different storage solutions and temperatures on hair follicle preservation. This randomized-controlled study included 10 androgenetic alopecia patients who underwent hair transplantation. Forty-five hair grafts were collected from each patient and then randomized to 8 different culture conditions for 7 days. Hair grafts were cultured in Williams' Medium E or Ringer's lactate solution (RLS) at either 4°C or 37°C, and with or without 10% PRP supplementation. In vitro hair growth in Williams' Medium E was significantly greater than in RLS. The 37°C temperature condition was found to be significantly better than the 4°C condition. The growth of hair grafts cultured with PRP was not significance difference from those without PRP. However, immunofluorescence staining for cytokeratin 15 showed greater expression in hair graft cultured with PRP. PRP may have a beneficial effect for preserving the viability of hair grafts. Williams' Medium E and 37°C temperature were found to be superior to RLS and 4°C relative to hair follicle growth in organ culture.

Fluoride Inhibits Longitudinal Bone Growth by Acting Directly at the Growth Plate in Cultured Neonatal Rat Metatarsal Bones.

Excessive intake of fluoride inhibits bone growth in both humans and animals. It is unknown whether fluoride acts directly on the growth plate to inhibit longitudinal bone growth, and its mechanism of action has not been elucidated. In this study, we used an organ culture system and SW1353 cells to evaluate the effects of fluoride on endochondral ossification. Neonatal rat metatarsal bones were dissected and cultured with or without fluoride for 7days. The total length and width of the metatarsal rudiments and the length of the calcification zone were measured. Chondrocyte proliferation, differentiation, and apoptosis were analyzed by immunohistochemistry and TUNEL assay in sectioned bones. The apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blotting in SW1353 cells. Linear measurements demonstrated that fluoride induced a biphasic effect on longitudinal bone growth in organ culture, with a significant growth inhibition at a high concentration (10-4M) and a stimulatory action at low concentration (10-6M) of fluoride. Histomorphometrical analysis of growth plate from fluoride-exposed metatarsal rudiments showed a significant reduction in the height of the proliferative and hypertrophic chondrocyte zones. Analysis of the Col2α1 and Col10α1 expression by immunohistochemistry revealed fluoride-suppressed metatarsal growth plate chondrocyte proliferation and differentiation. In addition, fluoride increased the number of apoptotic chondrocytes in the metatarsal growth plate. Western blotting showed an up-regulated expression of Caspase-3 and Bax and down-regulated expression of anti-apoptotic protein Bcl-2 after treatment with 5 × 10-4M fluoride in SW1353 cells. Our findings indicated that fluoride inhibited longitudinal bone growth by acting directly at the growth plate in cultured neonatal rat metatarsal bones. Such growth inhibition was mediated by suppressing proliferation and differentiation, increasing apoptosis of resting chondrocytes and causing premature cell senescence in the growth plate.

OR17-3 Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy particularly in severe, non-GH deficient conditions such as chondrodysplasias, and has potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we previously developed a cartilage-targeting single-chain human antibody fragment (CaAb) (1), aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. In the current study, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor-I (IGF-1), an endocrine/paracrine factor which positively regulates chondrogenesis. We first used ELISA to assess the ability of the CaAb-IGF1 fusion proteins to bind tissue lysates from different mouse organs and found that the fusion proteins bound to cartilage with high tissue specificity. We also found that these fusion proteins retain potent IGF-1 biological activity, as demonstrated by their ability to stimulate AKT and ERK phosphorylation in cell culture and stimulate metatarsal bone growth in organ culture. To assess in vivo efficacy, we administered daily subcutaneous injections to growth hormone-deficient (lit) mice for 5 days. In this model, twice-daily IGF-1 increased growth plate height but daily IGF-1 did not. In contrast, daily equimolar injections of the targeted fusion proteins did increase growth plate height whereas a fusion protein lacking the cartilage-targeting domain did not (P<0.01 targeted vs non-targeted or saline). Even alternate-day injections of the targeted fusion proteins were sufficient to produce a therapeutic effect (P<0.01) similar to daily injection. To assess off-target effects, we measured proliferation in kidney cortical cells by BrdU incorporation. Daily injections of the targeted fusion proteins did not increase kidney cell proliferation whereas twice-daily IGF-1 itself (the minimum IGF-1 regimen that stimulated the growth plate) did stimulate kidney proliferation (P<0.01). In summary, the fusion proteins, unlike IGF-I itself, were able to stimulate growth plate activity without significant off-target effects on kidney. Our findings therefore provide proof-of-principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders. Reference: (1) Cheung et al. Pharma Res 2015 Jul;32(7):2439-49. Sources of Funding: This work was supported by the Intramural Research Programs of NICHD and NCI, National Institutes of Health. Additional funding through Grant for Growth Innovation from EMD Serono, Inc. a biopharmaceutical business of Merck KGaA, Germany, and Endocrine Scholars Award in Growth Hormone Research from the Endocrine Society.

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

An Optimized Preparation Technique for Saphenous Vein Graft

An Optimized Preparation Technique for Saphenous Vein Graft

The neuropeptide galanin is a novel inhibitor of human hair growth

Galanin is a trophic factor of the central and peripheral nervous system that shows widespread distribution in human skin. However, the exact localization and the role of galanin in the hair follicle (HF) remain to be clarified. To characterize galanin expression in human scalp HFs and to examine the effects of galanin on normal human scalp HF growth in organ culture. Immunohistochemistry was performed on cryosections of human female scalp skin. Anagen HFs were microdissected and cultured up to 9 days and treated with 100 nmol L(-1) galanin. Staining for Ki-67, TUNEL and Masson-Fontana were used to analyse proliferation, apoptosis and hair cycle staging of the HFs. Functional effects of galanin were tested in serum-free HF organ culture. Galanin-like immunoreactivity was detected in the outer root sheath (ORS) and inner root sheath. Additionally, galanin mRNA was detected in ORS keratinocytes and all HF samples tested. Galanin receptor transcripts (GalR2, GalR3) were also detected in selected samples. Galanin reduced proliferation of hair matrix keratinocytes in situ compared with vehicle-treated controls, shortened the hair growth phase (anagen) in vitro and reduced hair shaft elongation. This was accompanied by the premature development of a catagen-like morphology of galanin-treated HFs. We present the first evidence that human HFs are both a source and a functionally relevant target of galanin. Due to its hair growth-inhibitory properties in vitro, galanin application deserves further exploration as a potential new treatment strategy for unwanted hair growth (hirsutism, hypertrichosis).

Polyamines and hair: a couple in search of perfection

Polyamines (spermidine, putrescine and spermine) are multifunctional cationic amines that are indispensable for cellular proliferation; of key significance in the growth of rapidly regenerating tissues and tumors. Given that the hair follicle (HF) is one of the most highly proliferative organs in mammalian biology, it is not surprising that polyamines are crucial to HF growth. Indeed, growing (anagen) HFs show the highest activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, while inhibition of ODC, using eflornithine, results in a decreased rate of excessive facial hair growth in vivo and inhibits human scalp hair growth in organ culture. In sheep, manipulation of dietary intake of polyamines also results in altered wool growth. Polyamine-containing nutraceuticals have therefore been proposed as promoters of human hair growth. Recent progress in polyamine research, coupled with renewed interest in the role of polyamines in skin biology, encourages one to revisit their potential roles in HF biology and highlights the need for a systematic evaluation of their mechanisms of action and clinical applications in the treatment of hair disorders. The present viewpoint essay outlines the key frontiers in polyamine-related hair research and defines the major open questions. Moreover, it argues that a renaissance in polyamine research in hair biology, well beyond the inhibition of ODC activity in hirsutism therapy, is important for the development of novel therapeutic strategies for the manipulation of human hair growth. Such targets could include the manipulation of polyamine biosynthesis and the topical administration of selected polyamines, such as spermidine.

Growth of the rat gubernaculum in vitro and localisation of its growth centre

Purpose Recent studies suggest that testicular descent is accomplished by outgrowth of the gubernaculum from the abdominal wall. The tip of the gubernaculum has been proposed as the primary site of growth, similar to an embryonic limb bud. We aimed to determine the maximum site of growth in organ culture. Methods Gubernacula from 1-day-old Sprague-Dawley rats (n = 40) were collected and divided into 4 groups as follows: whole gubernaculum (control), truncated gubernaculum (tip excised), gubernacular tip alone, and grafted gubernaculum with an extra tip on its side. Tissues were cultured with or without calcitonin gene-related peptide (CGRP) (714nmol/L) in medium for 24 hours. The area of each gubernaculum was determined by “Image J” analysis of digital photos collected via a Leica Wild M28 microscope (Leica Microsystems, Wetzler GmbH Germany) taken before and after culture. Results In organ culture, the neonatal rat gubernaculum normally shrank 10% to 15%, but this was prevented by the presence of exogenous CGRP (0.8% vs 11.8%; P < .003). By contrast, gubernacula with their tips excised were not affected by CGRP (3.4% vs 4.7%; not significant). Gubernacular tips alone did respond to CGRP (2.7% vs 13.5%; P < .03). Transplantation of the tip to another gubernaculum caused it to develop 2 tips. Conclusions These results suggest that the rat gubernaculum contains a growth centre in its distal tip that can respond to CGRP. This is consistent with a limb bud model of gubernacular growth during the inguinoscrotal descent of the testis.

Differentiation Associated Changes in Gene Expression Profiles of Interstitial Cystitis and Control Urothelial Cells

We evaluated gene expression profiles after inducing differentiation in cultured interstitial cystitis and control urothelial cells. Bladder biopsies were taken from patients with interstitial cystitis and controls, that is women undergoing surgery for stress incontinence. Primary cultures were grown in keratinocyte growth medium with supplements. To induce differentiation in some plates the medium was changed to Dulbecco's modified Eagle's minimal essential medium-F12 (Media Tech, Herndon, Virginia) with supplements. RNA was analyzed with Affymetrix(R) chips. Three patients with nonulcerative interstitial cystitis were compared with 3 controls. After inducing differentiation 302 genes with a described function were altered at least 3-fold in interstitial cystitis and control cells (p <0.01). Functions of the 162 up-regulated genes included cell adhesion (eg claudins, occludin and cingulin), urothelial differentiation, the retinoic acid pathway and keratinocyte differentiation (eg skin cornified envelope components). The 140 down-regulated transcripts included genes associated with basal urothelium (eg p63, integrins beta4, alpha5 and alpha6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins, and members of the Wnt and Notch pathways. When comparing interstitial cystitis control cells after differentiation, only 7 genes with a described function were altered at least 3-fold (p <0.01). PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased, and AKR1C2 and MKNK1 were increased in interstitial cystitis cases. Differentiation associated changes occurred in interstitial cystitis and control cells. Comparing interstitial cystitis vs control cases revealed few differences. This study may have included patients with interstitial cystitis and minimal urothelial deficiency, and/or we may have selected cells that were most robust in culture. Also, the abnormal urothelium in interstitial cystitis cases may be due to post-translational changes and/or to the bladder environment.

Urothelial Vascular Endothelial Growth Factor-a Expression in Fetal Bladder Outflow Obstruction

PURPOSE Vascular endothelial growth factor-A (VEGF-A) directs blood vessel formation in development and disease. Recently, it was demonstrated that VEGF-A enhanced embryonic bladder growth in organ culture, both in the urothelial and detrusor smooht muscle compartments. Therefore, we hypothesised that VEGF-A may play a role in diseases where bladder overgrowth occurs such as bladder outflow obstruction. MATERIAL AND METHODS VEGF-A expression was examined in the bladders of fetal sheep that had undergone short- (9 days) or long-term (30 days) bladder obstruction by combined urethral obstruction and urachal ligation at 75 days gestation. Short-term obstructuction leads to overgrowth of the whole bladder, resembling infants born with posterior urethral valves. When obstruction is maintained for 30 days, massively-dilated, thin walled bladders are poorly contractile, a phenotype resembling either the demcompensated bladder seen in some teenagers with posterior urthral valves or the prune belly syndrome. RESULTS VEGF-A was immunolocalised to the urothelium of the bladder in all experimental groups. In short-term obstruction, VEGF-A expression was upregulated versus time-matched controls and this group tended to have urothelium with multiple layers of nuclei. Conversely, animals obstructed for 30 days had patchy downregulation of VEGF-A expression in the urothelium which had become considerably thinner with few layers of nuclei. CONCLUSIONS VEGF-A is expressed in the fetal urothelium. There appears to be a biphasic response after bladder outflow obstruction and it is possible that increased VEGF-A plays a role in the actual growth response in this model.

Vascular Endothelial Growth Factor Mediates Hypoxic Stimulated Embryonic Bladder Growth in Organ Culture

Tissue hypoxia enhances embryonic angiogenesis at least in part by up-regulating vascular endothelial growth factor. Additionally, exogenous vascular endothelial growth factor-A enhances embryonic bladder explant growth. We hypothesized that developing bladders are hypoxic in vivo and oxygen tensions modulate explanted bladder growth by altering vascular endothelial growth factor-A expression. Embryonic day 14 mouse bladders were cultured in 20% O(2) or 3% O(2) atmospheres. Some cultures were supplemented with a vascular endothelial growth factor receptor 1/Fc chimera to block vascular endothelial growth factor bioactivity. After 6 days explant areas, DNA, protein, total cell numbers, and proportions expressing endothelial and smooth muscle markers were measured. Pimonidazole was administered to pregnant mice and hypoxia was sought in embryonic tissues by immunohistochemistry. In vivo pimonidazole adducts and vascular endothelial growth factor-A immunolocalized to embryonic urothelium and bladders up-regulated total vascular endothelial growth factor-A between embryonic days 14 and 18. All growth parameters and vascular endothelial growth factor-A protein levels were enhanced in hypoxic vs normoxic culture. Addition of vascular endothelial growth factor receptor 1/Fc prevented this accelerated growth. In vivo embryonic bladders are hypoxic and express vascular endothelial growth factor-A. In vitro, when oxygen tensions are manipulated, vascular endothelial growth factor-A protein positively correlates with the growth of whole explants as well as endothelium. Normal embryonic bladder development may be driven at least in part by hypoxic up-regulation of vascular endothelial growth factor-A.

Oocyte growth and ovarian tissue differentiation in organ culture of mouse fetal gonads

Mammalian oocytes undergo growth and development through prenatal and postnatal periods. This is an investigation of development in vitro of oocytes and ovarian tissues from fetal gonads of the 12.5 days post coitus. Cultured tissues survived and the oocytes entered the growth phase, subsequently achieving long-term (14-30 days) culture. Growing oocytes were observed in their general morphology and were enclosed within 1-2 layers of granulosa cells, which resembled primary or secondary follicles. However, no follicular structures were also found in the cultures despite the existence of growing oocytes. The oocytes enclosed in very flattened cells were detected as in histological structure on day 18 of culture. The oocyte diameter became approximately 50 μm under culture conditions supplemented with 15% fetal bovine serum or Ehrlich ascites tumor fluid. These results indicate that the maximum culture period for oocyte growth in this organ culture is approximately 3 weeks and that the oocytes can grow in vitro to at least a medium size even if normal follicular structure is not formed.

Role of Extracellular Matrix, Activin-A and Mitogen Activated Protein Kinase (MAPK) Signaling Pathways in Preantral Follicle Growth and Survival in the Mouse

We have previously shown that ECM-Activin interactions play role in preantral follicle growth in organ cultures (1). Our current objective was to investigate the role of extracellular matrix (ECM)-activin-A interactions in in-vitro growth and survival of mouse preantral follicles under different culture conditions, and to determine mitogen activated protein kinase (MAPK) signaling pathways in preantral follicle development. MAPK family consists of c-jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) and p38 which regulate cell growth, differentiation, and survival.

Growth, morphogenesis, and differentiation during mouse prostate development in situ, in renal grafts, and in vitro

In vitro organ culture and renal grafting of the urogenital sinus (UGS) have both been used as models of prostate development. However, neither has been rigorously examined for its fidelity to replicate the canonical process of prostate differentiation in situ. We assessed size, morphology, histology, and the mRNA expression of differentiation marker genes of the E14 male mouse UGS grown for 0-28 days as sub-renal capsule allografts in nude mice or in culture containing androgen and compared these to UGS development in situ. Development of grafted tissues was morphologically and histologically similar to development in situ but differentiation occurred more rapidly. UGS growth in organ culture resulted in bud formation, but did not trigger cellular differentiation. However, the potential for differentiation was maintained and could be rescued by grafting tissues into nude mice. In vitro organ culture and renal grafting of UGS tissues may be appropriate models for studying prostatic bud formation, but only grafting is an appropriate model for prostatic differentiation.

Oxidation–Reduction (Redox) Controls Fetal Hypoplastic Lung Growth

Introduction. The persistent morbidity and mortality of congenital diaphragmatic hernia are largely due to associated pulmonary hypoplasia. We have shown previously that three antioxidants (vitamin C, glutathione, and vitamin E) could accelerate the growth of fetal hypoplastic lungs grown in culture. We hypothesize that this occurs via a reductant mechanism. Methods. Timed-pregnant rats were gavage-fed nitrofen (100 mg) on day 9.5 of gestation (term = day 22). Fetal lungs were harvested on day 13.5 and placed in organ culture containing serum-free BGJb medium with antibiotics. After randomization, the lung organ cultures were divided into a control group ( n = 31) and an experimental group that received the antioxidant N-acetylcysteine (NAC, 100 μM, n = 31). The fetal lung organ cultures were grown for 4 days at 37°C with 5% CO 2. Computer-assisted digital tracings of the airways were performed daily on live, unstained specimens, and lung bud count, perimeter, and area were measured. After 4 days, lungs were pooled, homogenized, and assayed for reduced and oxidized glutathione, normalized to protein, as an estimate of the tissue redox potential. Data were expressed as means ± SEM, and statistical comparisons were performed using Student's unpaired t test, with P < 0.05 considered significant. Results. Area, perimeter, lung bud count, and complexity (as measured by the perimeter/square root of area) were all significantly increased with NAC treatment from day 2 onward. Reduced glutathione levels were significantly increased following NAC administration (67.1 ± 5.8 versus 37.5 ± 4.2 μmol/mg, P = 0.0004). The ratio of reduced to oxidized glutathione was 2.23. Conclusions. N-Acetylcysteine stimulates nitrofen-induced hypoplastic fetal lung growth in organ culture and increases the ratio of reduced to oxidized glutathione. These data support the concept that oxidation–reduction (redox) may be an important control mechanism for fetal lung growth.

Developmental and Hormonal Regulation of Transforming Growth Factor- 1 (TGF 1), -2, and -3 Gene Expression in Isolated Prostatic Epithelial and Stromal Cells: Epidermal Growth Factor and TGF Interactions

Growth factors are postulated to mediate stromal-epithelial interactions in the prostate to maintain normal tissue physiology. Transforming growth factor-β (TGFβ) has been shown to influence the prostate and probably mediate stromal-epithelial interactions. TGFβ1 messenger RNA (mRNA) expression is stimulated after castration and can be suppressed by in vivo treatment with androgens. The hypothesis tested is that TGFβ is regulated not only by androgen, but also by a network of locally produced growth factors that influence prostatic growth and differentiation. Epithelial and stromal cells from 20-day-old rat ventral prostate were isolated and used to test this hypothesis. The expression of mRNA for TGFβ1, -2, and -3 was analyzed by a quantitative RT-PCR procedure. Observations from this assay demonstrate that both epithelial and stromal cells express the mRNA for TGFβ1, -2, and -3. TGFβ1 mRNA expression was constant during development of the prostate. TGFβ2 mRNA expression was elevated at birth, then declined and elevated again at 100 days of age. TGFβ3 mRNA expression was high during puberty and young adult ages then declined at 100 days of age. TGFβ2 and TGFβ3 expression are inversely related during prostate development. After castration of 60-day-old rats, both TGFβ1 and TGFβ2 mRNA were enhanced. Interestingly, TGFβ3 mRNA was significantly suppressed after castration. Epidermal growth factor (EGF) stimulated TGFβ1 mRNA expression in stromal cells (6-fold increase), whereas keratinocyte growth factor stimulated TGFβ2 mRNA in epithelial cells. TGFβ inhibited both testosterone- and EGF-stimulated prostatic stromal and epithelial cell growth. EGF and TGFβ also inhibited prostatic ductal morphogenesis and growth in organ culture. Immunocytochemical localization of TGFβ in 20-day-old prostate demonstrated predominately stromal localization of the protein. These results indicate that the isoforms of TGFβ2 and TGFβ3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFβ expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFβs are regulated indirectly through a complex network of growth factors, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be in part mediated by the increase in TGFβ1 expression. In summary, a network of growth factor-mediated stromal-epithelial interactions is needed to maintain prostate growth and development. TGFβ is postulated to have an important role in this process.

Developmental and hormonal regulation of transforming growth factor-beta1 (TGFbeta1), -2, and -3 gene expression in isolated prostatic epithelial and stromal cells: epidermal growth factor and TGFbeta interactions.

Growth factors are postulated to mediate stromal-epithelial interactions in the prostate to maintain normal tissue physiology. Transforming growth factor-beta (TGFbeta) has been shown to influence the prostate and probably mediate stromal-epithelial interactions. TGFbeta1 messenger RNA (mRNA) expression is stimulated after castration and can be suppressed by in vivo treatment with androgens. The hypothesis tested is that TGFbeta is regulated not only by androgen, but also by a network of locally produced growth factors that influence prostatic growth and differentiation. Epithelial and stromal cells from 20-day-old rat ventral prostate were isolated and used to test this hypothesis. The expression of mRNA for TGFbeta1, -2, and -3 was analyzed by a quantitative RT-PCR procedure. Observations from this assay demonstrate that both epithelial and stromal cells express the mRNA for TGFbeta1, -2, and -3. TGFbeta1 mRNA expression was constant during development of the prostate. TGFbeta2 mRNA expression was elevated at birth, then declined and elevated again at 100 days of age. TGFbeta3 mRNA expression was high during puberty and young adult ages then declined at 100 days of age. TGFbeta2 and TGFbeta3 expression are inversely related during prostate development. After castration of 60-day-old rats, both TGFbeta1 and TGFbeta2 mRNA were enhanced. Interestingly, TGFbeta3 mRNA was significantly suppressed after castration. Epidermal growth factor (EGF) stimulated TGFbeta1 mRNA expression in stromal cells (6-fold increase), whereas keratinocyte growth factor stimulated TGFbeta2 mRNA in epithelial cells. TGFbeta inhibited both testosterone- and EGF-stimulated prostatic stromal and epithelial cell growth. EGF and TGFbeta also inhibited prostatic ductal morphogenesis and growth in organ culture. Immunocytochemical localization of TGFbeta in 20-day-old prostate demonstrated predominately stromal localization of the protein. These results indicate that the isoforms of TGFbeta2 and TGFbeta3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFbeta expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFbetas are regulated indirectly through a complex network of growth factors, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be in part mediated by the increase in TGFbeta1 expression. In summary, a network of growth factor-mediated stromal-epithelial interactions is needed to maintain prostate growth and development. TGFbeta is postulated to have an important role in this process.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P < 0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enhancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l-1) significantly accelerated longitudinal femoral growth in organ culture (P < 0.05), whereas in vivo indomethacin (2 mg kg-1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral growth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Effects of 5‐Fluorouracil on embryonic rat palate in vitro: Fusion in the absence of proliferation

5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the present study, gestation day (GD) 14 embryonic rat craniofacial explants were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours, with a return to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at the high concentration, 2.0 micrograms/ml. After 5 days in culture, both DNA and protein decreased with increasing 5-FU concentration and duration of exposure. Also by the end of the culture period, 3H-TdR incorporation had decreased in a concentration dependent manner. It is concluded that progressive inhibition of proliferation and growth in organ culture results in two different morphological outcomes: cleft palate resulting from a narrowed oral cavity and increased incidence of anterior palatal fusion under conditions of strong growth reduction. This study demonstrates that elevation and fusion can occur in the absence of growth and proliferation. Based on these observations, severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.

IFN-gamma reverses IL-4 inhibition of fetal thymus growth in organ culture.

IL-4 is known to inhibit the growth and differentiation of 14-day-old fetal mouse thymus in organ culture. Here we report that IFN-gamma reverses this IL-4-mediated growth inhibition. Thymus lobes from 14-day-old fetuses were cultured for 12 days in medium containing 100 IU/ml rIL-4 either in the absence or presence of rIFN-gamma (100 to 1000 IU/ml). After culture, the cell yields and the absolute numbers and frequencies of the major subpopulations according the coordinate expression of CD4 and CD8 were estimated. IL-4 treatment alone was found to result in a seven-fold decrease in cell yield and an almost complete absence of the CD4+CD8+ subpopulation. Addition of IFN-gamma reversed IL-4-mediated inhibition in a dose-dependent fashion, with an optimal dose ranging from 200 to 500 IU/ml. IFN-gamma exerted this effect only when added within the first 48 h of initiating the culture. The specificity of the reversal effect was ascertained by neutralization of the effect by a neutralizing anti-IFN-gamma mAb and by lack of activity of human IFN-gamma. In the absence of IL-4, IFN-gamma had a growth-promoting effect as evident from a threefold increase in cell numbers.