Synthesis of platelet-derived growth factor by cells of splenic red pulp in normal rats

A population of cells in the spleens of normal rats was found to contain platelet-derived growth factor (PDGF) B chain mRNA. These cells were found predominantly in the red pulp and nuclear morphology of some was consistent with that of macrophages. Similar cells were also shown by immunocytochemical staining to contain PDGF-AB/BB. These PDGF-positive cells were also found almost exclusively in the red pulp. It has been suggested by others that PDGF plays an important role in the function of the lymphohemopoietic microenvironment.     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40–200m in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of >98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3–7.5 nM) or angiotensin II (0.001–1000 nM) induced ³H-thymidine incorporation and cell proliferation in a dose-dependent manner (P<0.01). S-nitroso-N acetylpencillamine (0.05–0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced ³H-thymidine incorporation (P<0.05) and cell proliferation (P<0.01). The antimitogenic effect of S-nitroso-N-acetylpencillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P<0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.     

Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide

Connexins are gap-junction proteins forming hexameric structures in the plasma membranes of adjacent cells, thereby creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. We have employed antisense oligonucleotides (AS) against rat CX43 to study the role of CX43 in dental pulp cells. Cultured dental pulp cells were treated with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was approximately 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS- and S-treated groups at 48 h (P < 0.01). An alkaline phosphatase assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, whereas S-treated pulp cells showed no marked changes. Western blot analysis revealed that heat-shock protein 25 was highly expressed in S-treated cells but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells highly expressed CX45, whereas S-treated cells exhibited high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization, and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns

Recent studies have documented that TGF-beta1 takes part in dental pulp tissue repair. Moreover, dental pulp cells have the potential to differentiate into odontoblast-like cells and produce reparative dentine in this process. However, the molecular mechanisms and potential interactions between TGF-beta1 and dental pulp cells are not clear due to the complexity of the pulp/dentine microenvironment. In this study, we investigated the induction of TGF-beta1 on the dental pulp cells in cell culture, tissue culture and three-dimensional culture patterns. These results demonstrated that TGF-beta1 significantly increased the proliferation of cells and activity of ALPase. Dental pulp cells cultured in the presence of TGF-beta1 formed mineralization nodules. In the organ culture, dental pulp cells treated with TGF-beta1 differentiated into odontoblast-like cells and formed a pulp-dentinal complex; and TGF-beta1 significantly induced synthesis of dentine relative proteins DSPP, DMP-1. The dental pulp cells share some characteristics of the odontoblast, such as a parallel arrangement with columnar form and a unilateral cell process. Together, these data indicate that TGF-beta1 can make dental pulp cells differentiated into odontoblast-like cells and form the pulp-dentinal complex. Moreover, these results suggest that TGF-beta1 is an important regulatory factor in odontoblast differentiation during tooth development and pulp repair.     

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor

Summary The weak base chloroquine and the Na⁺/H⁺ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12–16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 M and 5 M, respectively. To be fully active, the drugs have to be added within 4–8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [³H]leucine-labeled cellular protein and [¹²⁵I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0–8 h).     

Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. We have morphologically characterized the cells that produce obestatin and ghrelin in new-born and adult Sprague-Dawley rats that were freely fed, fasted, or subjected to gastric bypass surgery or reserpine treatment. Tissue samples collected from the gastrointestinal tract and pancreas were examined by double-immunofluorescence staining, immunoelectron microscopy, and conventional electron microscopy. Obestatin was present in the stomach, duodenum, jejunum, colon, and pancreas. In the stomach, differences were noted in the development of obestatin- and preproghrelin-immunreactive (IR) cells on the one hand and ghrelin-IR cells on the other, particularly 2 weeks after birth. Preproghrelin- and obestatin-IR cells were more numerous than ghrelin-IR cells in the stomach, suggesting the lack of ghrelin in some A-like cells. Most obestatin-producing cells in the stomach were distributed in the basal part of the oxyntic mucosa; these cells co-localized with chromogranin A (pancreastatin) and vesicle monoamine transporters type 1 and 2, but not with serotonin or histidine decarboxylase. Immunoelectron microscopy revealed the obestatin- and ghrelin-producing cells to be A-like cells, characterized by numerous highly electron-dense granules containing ghrelin and obestatin. Some granules exhibited an even electron density with thin electron-lucent halos, suggestive of monoamines. Feeding status, gastric bypass surgery, and reserpine treatment had no obvious effect on the A-like cells. In the pancreas, obestatin was present in the peripheral part of the islets, with a distribution distinct from that of glucagon-producing A cells, insulin-producing beta cells, and cells producing pancreatic polypeptide Y. Thus, obestatin and ghrelin co-localize with an anticipated monoamine in A-like cells in the stomach, and obestatin is found in pancreatic islets. This study was supported by a grant from the Cancer Foundation of St. Olav’s Hospital, Trondheim, Norway.     

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

Summary The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.     

Spatiotemporal distribution of insulin-like growth factor receptors during nephrogenesis in fetuses from normal and diabetic rats

Exposure to hyperglycemia in utero impairs rat nephrogenesis. The effect of maternal diabetes on insulin-like growth factors and their receptors in the fetal kidney is associated with an increase in both mRNA and protein of the insulin-like growth factor II/mannose 6-phosphate receptor. However, this receptor has never been localized in the fetal kidney. The spatial and temporal distribution of the three insulin-like growth factor receptors (insulin-like growth factor I receptor, insulin-like growth factor II/mannose 6-phosphate receptor and insulin receptor) in rat metanephros during both normal and streptozotocin-induced diabetic renal development was investigated using in situ hybridization and immunohistochemistry. All receptors were found in the fetal kidney from the start of nephrogenesis. Insulin-like growth factor I receptor expression was ubiquitous and continuously present during metanephric development. Insulin receptor expression was developmentally regulated during kidney maturation with an enhanced expression in proximal tubules at the late stages of development. Insulin-like growth factor II/mannose 6-phosphate receptor expression was ubiquitous in the early stages of development and was dramatically decreased at the late stages of normal kidney development. Insulin receptor and insulin-like growth factor I receptor expressions were unchanged in diabetic metanephroi. Although the spatial expression of insulin-like growth factor II/mannose 6-phosphate receptor was unaffected by hyperglycemia, its expression was not downregulated in the mesenchyme of the nephrogenic zone of diabetic fetuses on gestational day 20. This study suggests a crucial role of insulin-like growth factor II/mannose 6-phosphate receptor in the pathogenesis of the impaired nephrogenesis in fetuses of diabetic mothers.     

Histochemical and immunocytochemical study of hard tissue formation in dental pulp during the healing process in rat molars after tooth replantation

Dental pulp is assumed to possess the capacity to elaborate both bone and dentin matrix under the pathological conditions following tooth injury. This study was undertaken to clarify the mechanism inducing bone formation in the dental pulp by investigating the pulpal healing process, after tooth replantation, by micro-computed tomography (μ-CT), immunocytochemistry for heat-shock protein (HSP)-25 and cathepsin K (CK), and histochemistry for both alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Under deep anesthesia, the upper right first molar of 4-week-old Wistar rats was extracted and immediately repositioned in the original socket. In control teeth at this age, the periphery of the coronal dental pulp showed intense ALP-positive and HSP-25-positive reactions, whereas there were no TRAP-positive or CK-positive cells. Tooth replantation weakened or terminated ALP-positive and HSP-25-positive reactions in the pulp tissue at the initial stages. At 3–7 days after operation, the ALP-positive region recovered from the root apex to the coronal pulp followed by HSP-25-positive reactions in successful cases showing tertiary dentin formation. In other cases, TRAP-positive and CK-positive cells appeared in the pulp tissue of the replanted tooth at postoperative days 5–10 and remained associated with the bone tissue after 12–60 days. Immunoelectron microscopy clearly demonstrated that CK-positive osteoclast-lineage cells made contact with mesenchymal cells with prominent nucleoli and well-developed cell organelles. These data suggest that the appearance of TRAP-positive and CK-positive cells is involved in the induction of bone tissue formation in dental pulp.This work was supported in part by a grant from MEXT to promote 2001-multidisciplinary research project (in 2001–2005) and by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.).     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Irradiation-induced effects on the innervation of rat salivary glands: Changes in enkephalin- and bombesin-like immunoreactivity in ganglionic cells and intraglandular nerve fibers

When treating head and neck for cancer with the use of radiotherapy the salivary glands are usually within the treatment volume with ensuing dryness and discomfort. Since the autonomic nervous system is of pivotal importance for the salivary gland function and integrity, the irradiation-induced effects may involve an influence on the innervation of salivary glands. Therefore, the rat submandibular gland, including the submandibular ganglionic cells, has been subjected to immunohistochemical examination with respect to expression of neuropeptides following fractionated irradiation with high energy photons. A markedly enhanced expression of bombesin- and leu-enkephalin-(ENK)-like immunoreactivities (LI) in the ganglionic cells and a pronounced increase in the number of nerve fibers showing these immunoreactivities in the submandibular gland tissue following irradiation were observed 10 days after treatment. On the other hand, no changes in the patterns of VIP (vasoactive intestinal polypeptide)- and NPY (neuropeptide Y)-immunoreactivities occurred. Thus, the present study shows that alterations in the expression of certain neuropeptides take place in the submandibular gland and its associated ganglionic cells in response to irradiation of the head and neck region. These changes may add further explanation to the inherent radiosensitivity of salivary glands.     

Demonstration of uterine receptivity in vitro by co-culture of rat epithelial cells and blastocyst

Uterine receptivity is prerequisite for the attachment of the embryo to the uterine epithelium and involves a specialized polarity-dependent property of uterine epithelial (UE) cells. These UE cells, when polarized in culture, behave like cells in utero by exhibiting apico-basal polarity. In order to develop an implantation model in vitro, UE cells were polarized on extracellular matrix (ECM), and polarity was validated by response to estradiol-17β administered exogenously. UE cells of pregnant rats at day-3 and day-4 post-coitum (p.c.) and of non-pregnant rats were cultured on bare and extracellular-matrix-coated petri dishes until confluency. Hatched blastocysts were transferred to the cultures, and adhesion was monitored every 24 h. Although blastocysts attached to UE cells that were taken from non-pregnant rats and from rats of day-3 p.c. and cultured on bare plastic, they failed to attach to these cells polarized on ECM. However, blastocysts attached firmly to UE cells that had been taken from rats of day-4 p.c. and polarized on ECM. Receptivity of UE cells taken from non-pregnant and pregnant (day-4 p.c.) rats was quantitated by flow cytometric estimation of cellular levels of β3 integrin. The expression of β3 integrin in UE cells from rats of day-4 p.c. was highly significant (P<0.01) when compared with its expression in UE cells from non-pregnant rats. The expression of β3 integrin in UE cells of day-4 p.c. confirmed the receptivity of these cells to blastocyst implantation. Uterine receptivity was also validated in vivo by inducing the decidual cell reaction in rats ovariectomized on day-3 and day-4 p.c. Whereas remarkable deciduoma was noticed in the animals of day-4 p.c., it was absent in the animals of day-3 p.c., thereby indicating that the uterus was receptive on day-4 p.c. only. Thus, blastocysts do not attach to polarized UE cells that have been obtained from a non-receptive uterus. Attachment will occur only if the cells are obtained from a receptive uterus. UE cell receptivity is therefore essential for mimicking the process of implantation in vitro.The authors are grateful to the Ministry of Health and Family welfare, Government of India, for financial support