Platelet-derived growth factor levels in wounds of diabetic rats.

Delayed wound healing is a troublesome complication of Diabetes. Results from recent investigations concerning the potential cellular and molecular mechanisms responsible for diabetic wound healing deficiency are preliminary in nature. Some studies have demonstrated that direct application of certain growth factors/cytokines can facilitate wound healing in diabetic models. It is possible that refractory diabetic wounds are the result of deficiencies in growth factors/cytokines important for the normal wound healing process. Platelet-Derived Growth Factor (PDGF) levels were examined by radioimmunoassay in wound tissue of normal and diabetic rats (streptozotocin-induced diabetes). Immunohistochemical analysis was utilized to localize and characterize PDGF immunopositive cells at the wound site of normal and diabetic animals. At the wound site, normal animals demonstrated significantly elevated PDGF levels compared to diabetic animals at 5 days post-wounding (no differences were observed in the spleen or contralateral control tissue). There appeared to be a visible increase in PDGF immunopositive cells at the wound site in both experimental and control groups. By day 10 post-wounding, PDGF levels at the wound site in normal animals were reduced becoming similar to PDGF levels in diabetic animals. This corresponded to an apparent reduction of PDGF immunopositive cells in both groups (similar to baseline levels). PDGF levels in both groups remained stable until day 20 post-wounding when a significant elevation of wound site PDGF levels occurred in the diabetic group. The findings suggest that absence of an initial increase in PDGF may play an important role in poor wound healing observed in diabetic animals. The reduction in PDGF may be related to decreased cellular PDGF production rather than a lack of PDGF-producing cells. Perhaps the diabetic state inhibits cellular PDGF gene expression signaled by wounding or interferes with normal PDGF expression at the wound site.

Potentiation of haloperidol-induced catalepsy by ascorbic acid in rats and nonhuman primates.

Ascorbic acid was examined for potentiative effects on the catalepsy induced by haloperidol in rats and squirrel monkeys. In both animal species pretreatment with ascorbic acid (1000 mg/kg) markedly potentiated catalepsy induced by haloperidol. It is suggested that vitamin binds to, and inactivates, some brain dopamine receptors and in so doing potentiates an otherwise minimally cataleptogenic dose of haloperidol.

Comparison of behavioral effects of systemic L-DOPA and intracranial dopamine in mesolimbic forebrain of nonhuman primates.

The systemic administration of L-DOPA and carbidopa to six rhesus and four squirrel monkeys produced an initial period of depressed activity followed by increased locomotion, hypervigilance, involuntary oral-facial movements and a gnawing syndrome. The squirrel monkey exhibited a depressed phase, locomotor stimulation, searching behavior, stereotypic grooming and gnawing syndrome. Most of these activities were prevented by pretreatment with 0.1 mg/kg haloperidol. Bilateral injection of 100 microgram dopamine into the mesolimbic forebrain of four squirrel monkeys also produced an initial depression followed by hyperactivity similar to that produced by L-DOPA, but without gnawing. A stereotyped submissive or juvenile posturing occurred in three animals. These DA-induced activities were blocked by 0.1 mg/kg haloperidol. Similar injection of 100 microgram L-norepinephrine produced a profound depression followed by moderate activity coupled with loss of extensor muscle strength in the legs. Bilateral injection of 300 microgram dopamine into the nucleus accumbens of a rhesus monkey produced stereotypic pacing. These data confirm in primates the importance of dopaminergic mechanisms of the mesolimbic forebrain in locomotor activity and behavior.

Biphasic locomotor response to intra-accumbens dopamine in a nonhuman primate.

Locomotor activity of ten squirrel monkeys, Saimiri sciureus, was evaluated by means of a photocell activity cage following intracranial application of dopamine (DA). A biphasic response consisting of an initial quiet period followed by increased locomotor activity was seen following intra-accumbens DA, 12.5--100 micrograms bilaterally. Both the length of the quiet phase and intensity of locomotor activity were positively related to DA dose. Intra-caudate DA (50 micrograms) was significantly less effective in producing locomotor effects. The specificity of the DA response was substantiated by dose-related inhibition with both systemic (0.1 or 0.05 mg/kg) and intra-accumbens (2--10 micrograms) administration of the DA antagonist haloperidol. Additionally, the intra-accumbens application of haloperidol was found to be ineffective in inducing catalepsy, a state readily produced by systemically administered haloperidol.

Myofibroblasts in phenytoin-induced hyperplastic connective tissue in the rat and in human gingival overgrowth.

Phenytoin is a commonly used anticonvulsant drug for the prevention of seizures. A common side effect of phenytoin (PHT) therapy is connective tissue hyperplasia, particularly in the oral cavity manifesting as gingival overgrowth. Our previous studies concerning the molecular mechanisms of drug-induced gingival overgrowth have demonstrated that PHT alters the normal tissue turnover/wound healing signal by causing changes in macrophage phenotype, resulting in the upregulation of essential polypeptide growth factors such as platelet-derived growth factor (PDGF). The cellular consequences of this elevation in growth factor have not been investigated. The present light and electron microscopic study of rat hyperplastic connective tissue and human gingival overgrowth induced by PHT treatment revealed the presence of numerous myofibroblasts. Cells identified as myofibroblasts were evident in all PHT-treated tissue samples and were characterized by an elongated fusiform cell shape, abundant cytoplasmic rough endoplasmic reticulum/polyribosomes, and accumulations of sub-plasmalemmal microfilaments containing spindle densities. These cells were never observed in control tissues. Myofibroblasts are associated with the later stages of tissue turnover, specifically with the transition from the granulation to the remodeling phases of the wound healing process. The presence of myofibroblasts in hyperplastic connective and gingival tissues induced by PHT treatment suggests that PHT exacerbates the normal tissue turnover/wound healing signals responsible for the appearance of myofibroblasts.

Quantitation of phagocytic cells in phenytoin-induced connective tissue proliferation in the rat.

This study demonstrates that, in rats, systemically administered phenytoin produces a statistically significant increase in accumulation of phagocytic and esterase positive cells in areas of phenytoin-induced connective tissue proliferation. Many of these cells are perceived to be macrophages. Since macrophages have been shown to produce chemoattractant and mitogens for fibroblasts, they may very well play a major role in the gingival hyperplasia seen in patients taking phenytoin. The results of the study show that the effects of phenytoin on fibroblasts may be more complex than previously thought.

Cyclosporine A upregulates platelet-derived growth factor B chain in hyperplastic human gingiva.

Cyclosporine A (CSA) is a widely used immunosuppressant for transplant patients and is also used for the treatment of a wide variety of systemic diseases with immunologic components. A prominent side effect of CSA administration is gingival overgrowth (hyperplasia). It has been postulated that CSA alters fibroblast activity through effects on various growth factors/cytokines. However, as yet, data concerning the molecular mechanisms involved in pathologic connective tissue proliferation are preliminary in nature. Our previous investigations concerning phenytoin-induced effects on platelet-derived growth factor B (PDGF-B) gene expression have demonstrated that other drugs which cause gingival overgrowth can upregulate macrophage PDGF-B gene expression in vitro and in vivo. The purpose of the present study was to evaluate PDGF-B gene expression in gingival tissues of patients receiving CSA therapy and exhibiting gingival overgrowth to determine if similar PDGF-B upregulation occurs in response to CSA and to identify PDGF-B producing cells in these tissues. Quantitative competitive reverse transcription polymerase chain reaction (QC-RTPCR) techniques were utilized to measure PDGF-B mRNA levels in CSA overgrowth patients and normal controls (N = 6/group). Results were expressed as mean +/- mRNA copy number and tested for significance using unpaired t-tests. Gingival samples were harvested (standardized for local inflammation at the sample site), total RNA was extracted, and QC-RTPCR was performed using specific PDGF-B primers and a corresponding competitive internal standard. CSA-treated patients exhibiting gingival overgrowth demonstrated approximately 48-fold increase in PDGF-B mRNA (7667.1 +/- 477.4 copies for CSA patients vs. 158.2 +/- 37.1 copies for controls; P < 0.001). Additionally, dual fluorescence immunohistochemistry for mature macrophage marker antigen (CD51) and intracellular PDGF-B was utilized to identify and localize PDGF-B producing cells were demonstrated to be macrophages distributed in a non-uniform manner throughout the papillary connective tissue. These results further support the hypothesis that the molecular mechanisms responsible for drug-induced gingival overgrowth may involve upregulation of PDGF-B macrophage gene expression. We continue to investigated specific CSA-induced alterations of macrophage PDGF-B gene expression in vitro and in vivo.

Phenytoin increases gene expression for platelet-derived growth factor B chain in macrophages and monocytes.

The mechanism by which phenytoin (PHT) induces gingival overgrowth remains unclear. We hypothesized that PHT increases macrophage production of platelet-derived growth factor (PDGF), an important cytokine in connective tissue growth and repair, and that excessive production PDGF in gingiva could lead to redundant growth. To test the hypothesis, rat peritoneal macrophages and human blood monocytes were cultured in the presence of PHT (5 to 20 micrograms/ml medium) or an equal volume of its solvent for 3 days and tested for expression of PDGF-B mRNA by in situ hybridization. Approximately 300 cells/culture well were examined (3 wells/drug level) for positive indication of PDGF-B mRNA. Data were compared by chi square test. All levels of PHT in both cell types induced a 2- to 8-fold increase in PDGF-B mRNA positive cells, significant in all cases at P < 0.001. Northern blot analysis of RNA from similarly cultured rat macrophages confirmed these findings. Cells treated with 10 micrograms PHT/ml medium or solvent revealed 2.2 +/- 0.3 and 1.0 +/- 0.2 (mean +/- SEM) arbitrary units PDGF mRNA respectively (t tests, P < 0.05). Additionally, rat macrophages were cultured in presence of 5 micrograms PHT/medium or its solvent and medium was analyzed for PDGF secretion by radioimmunoassay. Mean values (+/- SEM) were 1.28 +/- 0.49 and 0.78 +/- 0.07 ng/mg protein respectively (t test, P < 0.05). These data showed that PHT augmented the expression of c-sis, the gene for PDGF-B, and offered a possible explanation for PHT-induced gingival overgrowth.

PDGF-B producing cells and PDGF-B gene expression in normal gingival and cyclosporine A-induced gingival overgrowth.

It has been proposed that healthy gingiva is in a continuous state of wound repair. Thus, one might expect to find cells in normal gingiva producing growth factors associated with wound healing such as platelet-derived growth factor B chain (PDGF-B). One might also expect to find increased numbers of these cells or increased amounts of these growth factors in conditions which involve increased tissue volume such as drug-induced gingival overgrowth (DGO). The purpose of this study was to quantify PDGF-B gene expression and identify cells producing PDGF-B in normal gingiva and DGO. Cyclosporine A (CSA) was selected as a prototype of the overgrowth condition. Twelve patients with clinical CSA DGO and 12 patients with no DGO or history of drugs known to cause DGO were selected for study. Frozen sections of gingival specimens from these patients were subjected to in situ hybridization for PDGF-B mRNA. Positive cells were counted and expressed as mean +/- SEM cells/mm2 of lamina propria. Morphometric analysis revealed 6.2 +/- 1.9 cells/mm2 for control gingiva and 10.3 +/- 3.4 cells/mm2 for CSA DGO samples. There was no statistically significant difference between groups. PDGF-B gene expression was measured in these cells and expressed as mean +/- SEM silver grains/cells. There was a significant upregulation of PDGF-B gene expression in cells from the CSA DGO group (39.5 +/- 14.7 silver grains/cell for normal gingiva vs. 255.3 +/- 77.1 silver grains/cell for CSA DGO samples; P < 0.001). The presence of PDGF-B in these cells was confirmed in all cases by immunocytochemical localization. Additionally, PDGF-B producing cells were identified as macrophages in sections taken from an additional patient with CSA DGO by double immunofluorescence labeling of the CD51 membrane marker for macrophages and intracellular PDGF-B. These findings are consistent with the concept that healthy gingiva is in a continuous state of wound repair and support the hypothesis that CSA DGO is associated with enhanced macrophage PDGF-B gene expression rather than an increase in the number of PDGF-B producing macrophages.

Phenytoin and cyclosporine A specifically regulate macrophage phenotype and expression of platelet-derived growth factor and interleukin-1 in vitro and in vivo: possible molecular mechanism of drug-induced gingival hyperplasia.

Phenytoin (pht) is an anticonvulsant drug commonly used for the prevention of seizures. A common side effect of PHT therapy is gingival hyperplasia, occasionally so severe that it requires surgical intervention. Cyclosporine A (CSA) is a drug widely used for the control of rejection phenomena following solid organ and bone marrow transplantation. A frequent side effect of CSA administration is gingival overgrowth. As yet, the molecular mechanisms of drug-induced gingival hyperplasia are unknown although it has been postulated that certain drugs increase fibroblastic activity through alterations in levels of various growth factors and cytokines. The purpose of this study was to: 1) evaluate monocyte/macrophage platelet-derived growth factor (PDGF) and interleukin (IL)-1 beta production in vitro after exposure to CSA; 2) determine the levels of PDGF-B and IL-1 beta gene expression in minimally inflamed gingival tissues of control patients and PHT-treated patients exhibiting gingival overgrowth as well as patients with severe gingival inflammation; and 3) combine characterization of macrophage phenotype with clinical presentation and expression of PDGF-B and IL-1 beta in gingival tissues from the control and PHT-treated patients. For the in vitro studies, commercial ELISA kits were used to measure PDGF-A/PDGF-B and IL-1 beta levels in conditioned media from rat and human monocyte/macrophage cell cultures. CSA caused a significant elevation of PDGF but did not cause any changes in IL-1 beta levels. For the in vivo studies, quantitative competitive reverse transcription polymerase chain reaction (QC-RTPCR) techniques were utilized to measure PDGF-B and IL-1 beta mRNA levels in experimental groups. PHT-treated patients exhibiting gingival overgrowth demonstrated a significant increase in PDGF-B mRNA compared with minimally inflamed controls. Patients with severe gingival inflammation also demonstrated a significant increase in PDGF-B mRNA however, PHT-induced PDGF-B upregulation is approximately 6 times larger than PDGF-B upregulation produced by inflammation alone. PHT-treated patients exhibiting gingival overgrowth demonstrated no significant increase in IL-1 beta mRNA; however, IL-1 beta mRNA levels in the severely inflamed gingival samples demonstrated a significant increase. Additionally, for the clinical samples, macrophage phenotype was characterized immunohistochemically in adjacent sections using specific monoclonal antibodies for inflammatory and reparative/proliferative phenotypes. There were no significant differences in the numbers of either macrophage phenotype in minimally inflamed gingival tissues; however, in the severely inflamed tissue, there was a significant increase in the inflammatory macrophage phenotype and in the hyperplastic gingival tissue, there was a significant increase in the reparative/proliferative macrophage phenotype. The results of this investigation indicate that the clinical presentation of inflamed and hyperplastic gingival tissues is associated with specific macrophages phenotypes which express the pro-inflammatory cytokine IL-1 beta in inflamed tissues or the essential polypeptide growth factor PDGF-B in PHT-induced hyperplastic tissues.

Analgesic effects of 3-methoxybenzamide in rats.

The i.p. injection of 3-methoxybenzamide (3-MBA) in rats produces a dose-related elevation of the threshold for response to a painful stimulus. Metoclopramide, also a substituted benzamide, has analgesic activity that is attenuated by bromocriptine, a dopamine receptor agonist, and by the narcotic antagonist, naloxone, suggesting involvement of dopamine and opiate receptors in the action of this drug. The involvement of these receptors in the analgesic action of 3-MBA has been examined using L-dopa and naloxone. Neither significantly altered the analgesic action. Although the results are preliminary, the analgesic action of 3-MBA would not seem to occur via opiate or dopamine receptors.