Natriuretic peptide C receptor (NPR-C) expression in rat mesangial cells is downregulated by platelet-derived growth factor (PDGF) and the protein kinase C agonist phorbol myristate acetate (PMA). This study shows that PDGF and PMA diminish NPR-C mRNA abundance and that PMA does so by accelerating the degradation of the transcript. Exposure to PMA (0.1 microM) decreased mesangial cell NPR-C mRNA levels by more than 50% within 3 h and 125I-atrial natriuretic peptide binding by approximately 50% within 6 h. Disappearance of NPR-C transcripts after PMA treatment was more than twice as rapid as that seen after inhibition of RNA transcription with actinomycin D. Treatment with PDGF A/B (10 ng/ml) also produced downregulation of NPR-C mRNA, but the rate of transcript disappearance was similar to that seen after actinomycin D. Coincubation with actinomycin D inhibited the rapid disappearance of NPR-C mRNA with PMA. NPR-C mRNA levels increased four- to eightfold within 6 h after treatment with the protein synthesis inhibitor cycloheximide, but simultaneous treatment with PMA or PDGF still decreased the level of NPR-C mRNA despite the presence of cycloheximide. These results indicate that NPR-C expression is rapidly regulated by changes in the rate of catabolism of its mRNA through a protein kinase C-activated mechanism that depends on transcription. Treatment with cycloheximide induces NPR-C mRNA, but downregulation of this mRNA by either PDGF or PMA does not depend on synthesis of new protein.

Recent evidence suggests that bradykinin (BK) plays a role in regulating neointimal formation after vascular injury. The present study examined the mechanism whereby BK regulates platelet-derived growth factor (PDGF) AB-induced mitogenesis in smooth muscle cells from rat mesenteric artery. BK, but not other activators of phosphoinositidase C (e.g., angiotensin II), inhibited PDGF-stimulated mitogenesis. The B1 receptor agonist des-Arg9-BK (DABK) was more potent than the B2 agonist BK; smaller BK fragments had no activity. In studies in which the B2 receptor antagonist HOE-140 {D-Arg0[Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oic 8]BK} and the B1 receptor antagonist DHOE [[D-Arg0,Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oi c8,des-Arg9]BK] were used, both receptors independently mediated inhibition of PDGF-induced mitogenesis. There was no evidence for metabolism of BK to DABK. The rank potency for activating phosphoinositidase C and increasing intracellular Ca2+ (BK>> DABK) was opposite that for inhibiting mitogenesis (DABK>> BK). Inhibition of cyclooxygenase did not prevent the kinin-mediated inhibition. Kinetic analysis of the cell cycle effects of kinins on PDGF-stimulated mitogenesis revealed that continuous exposure to DABK or BK was inhibitory even when added shortly before the cells initiated DNA synthesis (S phase). However, short-term exposure (5-60 min) to DABK or BK was inhibitory only when added after exposure to PDGF. These data suggest that the B1 and B2 receptors potently inhibited PDGF-stimulated mitogenesis and proliferation by activating an alternative signal transduction cascade not involving phosphoinositidase C or prostaglandins. The inhibition occurred at a point late in progression of the cell cycle from G1 to S and was dependent on the presence of kinins after exposure to PDGF.

BACKGROUND

Human platelet concentrates (PCs) may be a source material to produce purified growth factors (GFs) for clinical use or cell therapy. However, no fractionation process of therapeutic-grade GF from PCs has ever been developed.

METHODS

PCs were virally inactivated by solvent/detergent (S/D) treatment, subjected to oil extraction to remove part of the S/D agents, and fractionated on a SP-Sepharose (SP) chromatographic column equilibrated in a phosphate-buffered saline (PBS) buffer, pH 7.5. The breakthrough was recovered, and the column was washed with the PBS buffer and then eluted by a 0.7 mol/L NaCl-PBS buffer pH 7.5 (SP-eluate). The SP-breakthrough and SP-eluate were characterized for their content in GF, proteins, lipids, and S/D agents. The MTS value of three cell lines cultivated in a medium containing 10% fetal bovine serum supplemented with 1% to 3% of SP-eluate or recombinant human (rHu) platelet-derived growth factor (PDGF)-BB was compared.

RESULTS

The SP-eluate contained a mean of 47, 17, and 6 ng/mL PDGF-AB, -BB, and -AA, respectively, and 0.26 ng/mL vascular endothelial growth factor (VEGF). It was largely depleted of transforming growth factor-β1 (2.33 ng/mL), epidermal growth factor (0.09 ng/mL), insulin-like growth factor (3.40 ng/mL), albumin, immunoglobulin (Ig)G, IgM, IgA, and fibrinogen, which were mostly in the breakthrough. tri-n-butyl phosphate and Triton X-45 were less than 2 ppm. Cell growth-promoting activity of the SP-eluate was at least as good as that of rHu-PDGF-BB.

CONCLUSIONS

Human PC can be fractionated into a purified, virally inactivated PDGF and VEGF concentrate, opening perspectives for the development of a new range of blood products for clinical use and cell therapy procedures.

Although growth cones typically collapse after encountering O1/galactocerebroside (GalC)-positive oligodendrocytes, the majority of growth cones traversed oligodendrocytes, which were raised for 8-10 d in medium containing 10 ng/ml platelet-derived growth factor (PDGF). Oligodendrocytes raised 8-10 d in control medium caused growth cone collapse as they normally do, but failed to elicit this response after being transferred to PDGF-containing medium for an additional 8-10 d. The opposite was observed when PDGF-treated oligodendrocytes were brought to control medium. Growth cones collapsed when contacting these cells. Oligodendrocytes also lost their collapse-inducing activity when raised in medium conditioned by astrocytes, known to produce PDGF. Antibody IN-1 is directed against against neurite growth inhibitors (NI), proteins of 35 and 250 kDa on the surface of O1/GalC-positive oligodendrocytes, which are known to elicit growth cone collapse. IN-1 immunoreactivity was markedly reduced in PDGF-treated oligodendrocytes. However, both PDGF-treated and control oligodendrocytes exhibited myelin-associated glycoprotein, proteolipid protein, and myelin basic protein immunoreactivity. This suggests that PDGF-treatment affects NI expression but does not interfere with the expression of advanced myelin marker proteins. Because NI cause growth cone collapse, the loss of collapse-inducing activity by PDGF-treated oligodendrocytes suggests that PDGF regulates, directly or indirectly, the expression of these proteins.

OBJECTIVE

To observe the effects of paeoniflorin on the expressions of CTGF and PDGF in liver tissue of fibrosis and the serum level of TNF-alpha in mice infected with Schistosoma japonicum, and to explore the protective effect and its mechanisms of paeoniflorin on liver fibrosis.

METHODS

Kunming mice were divided randomly into 5 groups, namely normal control group (Group A), paeoniflorin groups (Group B, C, D) and infected control group (Group E). The mice in Group B-E were infected with cercariae of Schistosoma japonicum, and then they were treated with praziquantel (400 mg/kg per day) for 2 days after 6 weeks. After that, the mice in Group B, C, D were given paeoniflorin with a dose of 30, 60, 120 mg/(kg x d), respectively. After 8 weeks of paeoniflorin treatment, all the mice were killed, and their livers and serum were obtained. Hematoxylin and eosin stain and Masson stain were used to observe the degree of hepatic fibrosis. Immunohistochemical staining was performed to detect the expressions of CTGF and PDGF in liver tissue. The serum level of TNF-alpha was detected by ELISA.

RESULTS

The expression levels of CTGF and PDGF proteins in liver tissue and the serum level of TNF-alpha of the mice in the high dosage paeoniflorin treatment group (Group D) were significantly lower than those in the infected control group (P < 0.05).

CONCLUSIONS

The effects of paeoniflorin on hepatic fibrosis induced by Schistosoma japonicum infection depends on its dosage. Paeoniflorin may exert its effects by inhibiting the serum level of TNF-alpha and down regulating the expression of CTGF and PDGF proteins.

OBJECTIVE

To determine whether nuclear transcription factor-kappaB (NF-kappaB) could modulate the expression of platelet-derived growth factor (PDGF) in rat retina induced by interleukin-1beta (IL-1beta) and studied the mechanism of inflammation-associated retinal diseases such as intraocular inflammation and proliferative vitreoretinopathy (PVR).

METHODS

One hundred and twenty eight Sprague-Dawley rats were divided into A, B, C and D groups, the right eyes were served as experimental eyes, and the left eyes as normal controls. Animals received 2 intravitreal injections at 1 hour interval, which included BSS and BSS (group A); BSS and IL-1beta (group B); pyrrolidine dithiocarbamate (PDTC) and BSS (group C); PDTC and IL-1beta (group D). PDTC was one of special inhibitors of NF-kappaB activation. The Rat eyes were examined by slit-lamp before intravitreal injection, 4 and 24 hours after the injection. Degree of intraocular inflammation was graded using a scoring system. Rats were sacrificed 4 and 24 hours after the injection, the expressions of NF-kappaB and PDGF were detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTS

Four hours after the injection, eyes in group B, as compared with group D, showed more severe intraocular inflammatory signs such as photophobia, iris hyperemia, miosis, large amount of fibrin exudates and posterior synechia of iris. Histopathologic examination showed numerous inflammatory cells within the retinal parenchyma, vitreous and ciliary body. Positive cells of NF-kappaB and PDGF in group D, which could be observed within inner nuclear layer and ganglion cell layer, were obviously less than that in group B. The expression of PDGF mRNA was less in group D compared with group B (P < 0.05), however the expression of PDGF-A mRNA was significantly higher than PDGF-B in group B and group D (P < 0.05). Twenty four hours after the injection, intraocular inflammation alleviated gradually, the expression of PDGF and NF-kappaB decreased obviously.

CONCLUSIONS

These results suggest that NF-kappaB modulates the expression of PDGF in rat retina induced by IL-1beta. Upregulation of PDGF may play an important role in the occurrence of intraocular inflammation and the different isoform of PDGF may have different impact on various ocular diseases.

Chronic liver disease evaluation is a very complicated process requiring complex assessment of numerous liver functions. In addition to standard methods of investigation we perform biotransformation liver tests for evaluation of microsome enzyme system. Markers of fibrogenesis represent modern noninvasive tests for fibrotic liver process detection in different diseases. The key role in the process of fibrogenesis have the adipose liver cells (ITO cells) producing collagen I, III, IV and lamilin. These cells may be transformed into myofibroblasts-like cells under specific conditions. Kupffer cells and monocytes produce substances stimulating the proliferation and transformation of liver ITO cells as also proteoglycans and hyaluronic acid synthesis. Mediators of this fibrogenetic activity are platelet derived growth factor (PDGF), transforming growth factors alpha and beta, lymphokines and monokines released by T-lymphocytes and macrophages, interleukin 1-alpha and interferon-tau. Acetaldehyde and its metabolites are important stimulators of collagen production by liver fibroblasts. The most often used markers of hepatic fibrogenesis are the following: procollagen III peptide, procollagen IV. type (one of its end carboxypeptide chains is determined-either with 7s collagen or NC1), hyaluronic acid, fibronectin, tenascine and unduline. As the most sensitive markers of fibrinogenesis are considered: hyaluronic acid, laminine, procollagen IV. type. Less often used are enzymes participating in collagen synthesis: prolyl-4-hydroxylase,lysyl-hydroxylase, galactosyl-hydroxylysyl-glucosyl-transferase, monoaminooxidase and N-acetyl-beta-D-glucoseaminidase. Breakdown of collagen is a multienzymatic process, catalysed by collagenases and other proteolytic enzymes. Decreased activity of collagenase is a supporting factor of cirrhosis development. Cirrhosis may be connected also with the levels of inhibitors such as e.g. serum/tissue? inhibitor of metalloproteinase. Biochemical markers of fibrogenesis are useful in regular monitoring of disease development and treatment effectivness and should be an inseparable part of progression assessment in all chronic hepatopathies. (Fig. 3, Ref. 49.)

There is a lack of information about the methods used for bovine platelet-rich plasma (PRP)/platelet-rich gel (PRG) procurement, including information on platelet (PLT), white blood cell (WBC) in PRP, and growth factor release from PRG supernatants. The aims of this study were to compare and to correlate the PLT, WBC, transforming growth factor beta-1 (TGF-β1), and platelet-derived growth factor BB (PDGF-BB) concentrations in bovine whole blood, plasma, and four PRP layers and their respective PRG supernatants: A and B (obtained by a single centrifugation tube method at 720g/5 min) and C and D (obtained by a double centrifugation tube method, by using two centrifugation episodes at 720g/5 min). PLT and WBC counts were significantly higher in PRP-C, followed by whole blood, PRP-A, PRP-B, and PRP-D. TGF-β1 concentrations were significantly higher in PRG-B supernatants and its correspondent PRP-B lysate when compared to the other PRG supernatants and plasma. Supernatants from PRG-A, PRG-B, and PRG-D had equivalent TGF-β1 concentrations. PDGF-BB concentrations were not statistically different between the hemoderivatives. Significant Pearson correlations were noted between PLT counts and WBC counts (0.8) and between PLT counts and PLT distribution width (0.6). Further studies should be performed to assess the potential clinical applications of these PRPs.

MC (mesangial cell) proliferation is closely linked to the progression of glomerular disease. It has been reported that cAMP effectors suppress MC proliferation, inhibiting activation of MAPK (mitogen-activated protein kinase). In fibroblasts, activation of MAPK induces the expression of type <em>D</em> cyclin, whereas, in MCs, this induction has not been shown. In the present study, we explored the effects of cAMP on MAPK and expression of cell-cycle-regulated proteins. <em>PDGF</em> (platelet-derived growth factor) stimulated MAPK activity, up-regulated protein levels of cyclin <em>D</em>1, C<em>D</em>K2 (cyclin-dependent kinase 2) and PCNA (proliferating cell nuclear antigen), decreased the protein level of p27 and increased <em>D</em>NA synthesis. Fsk (forskolin) or P<em>D</em>98059 suppressed <em>PDGF</em>-induced <em>D</em>NA synthesis. Both agents inhibited <em>PDGF</em>-stimulated mRNA and protein expression of cyclin <em>D</em>1 and C<em>D</em>K2. Fsk or P<em>D</em>98059 also inhibited protein expression of PCNA and blocked a decrease in p27 protein. Fsk induced the phosphorylation of Raf-1 at Ser259, which was inhibited by KT5720. These data suggest that cAMP inhibits MC proliferation through inhibition of MAPK activity, and this mechanism partly involves alteration in the levels of cell-cycle-regulated proteins.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRalpha, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin DDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

Since little is known about the function of polypeptide growth factors as regulators of multiple cell cycles, we compared the ability of FGF1, PDGF-AB and serum to induce a second round of DNA synthesis in Swiss 3T3 cells previously exposed to either FGF1, PDGF-AB or serum during the first cell cycle using [14C]- and [3H]thymidine in a double labeling system to distinguish between the first and second cell cycles. Surprisingly, we observed that cells exposed to either FGF1 or PDGF-AB in the first cell cycle were unable to synthesize DNA in response to FGF1 or PDGF-AB in the second cell cycle; yet these cells responded well to serum as a second cycle mitogen. Interestingly, while cells exposed to either FGF1 or PDGF-AB in the second cycle displayed normal receptor-mediated signaling and expressed cyclin D and E, they, like senescent fibroblasts and endothelial cells, failed to express cyclin A, and the continuous exposure of cells to either FGF1 or PDGF-AB resulted in a decrease in the kinase activity of the cyclin E/cdk2 complex. In addition, an increased association of this complex was observed with p21 CIP in an FGF1-dependent manner as well as with p27 KIP in a PDGF-AB-dependent manner. Lastly, the downregulation of p21 expression using an antisense strategy was able to partially rescue the replicative response of Swiss 3T3 cells to FGF1 in the second cycle. These data suggest that (i) FGF1 and PDGF-AB may limit their mitogenic effect to a single cell cycle, (ii) entry into the second round of replication is serum dependent and (iii) the self-limiting nature of FGF1 and PDGF-AB correlates with the accumulation of the cdk inhibitors, p21 and p27, respectively.

We determined the phospholipase D (PLD) activity in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKC-dependent mechanism of its activation. Depletion of calcium by EGTA and TMB-8 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calcium-dependent and independent mechanisms.

Increasing attention has been focused on the malignant tumor microenvironment, which plays important roles in tumor occurrence, progression and metastasis. Fibroblasts are recruited by platelet-derived growth factor (PDGFs) and invade the tumor microenvironment. In the PDGF family, PDGF-B has been reported to play an important role in the recruitment and invasion programs. However, whether PDGF-D plays a role in these programs remains unclear. We generated a recombinant plasmid expressing human PDGF-D and transfected the plasmid to dermal fibroblasts to examine the effects on cell invasive activities in 3D type I collagen gels. PDGF-D plasmid transfection enhanced fibroblast invasive activities both in invasive cell numbers and invasion depth in 3D collagen gels. These effects were blocked by Snail-specific siRNA transfection. PDGF-D transfection significantly induced Snail expression at both mRNA and protein levels. PDGF-D further upregulated MT1-MMP mRNA and protein expressions and this was inhibited when Snail was knocked down by siRNA. Both Snail and MT1-MMP expressions in fibroblasts and cellular invasive activities in 3D collagen induced by PDGF-D were inhibited by LY294002, SP600125, and U1026, the inhibitors of PI3K, JNK, and ERK1/2 signaling pathways, respectively. However, no effects were observed in response to the P38MAPK signaling pathway inhibitor SB203580. These effects of PDGF-D were confirmed by using the culture supernatants of the transfectants. Taken together, these data demonstrate that PDGF-D plays important roles in the recruitment and invasion programs of fibroblasts via the activation of PI3K, JNK and ERK1/2 signaling pathways, and upregulation of Snail and downstream effecter MT1-MMP. These findings indicate that PDGF-D is an important player in the tumor microenvironment for fibroblast recruitment.

Substantial evidence has demonstrated that platelet-derived growth factor-D (PDGF-D) is tightly associated with the development and progression of tumors. However, its biological functions in esophageal squamous cell carcinoma (ESCC) remain to be delineated. In this study, we found that expressions of PDGF-D mRNA and protein in ESCC tissues and cells were significantly higher than that in normal esophageal epithelial tissues (P < 0.05), further investigation showed that PDGF-D protein level in EC1 cells was obviously higher than those in EC9706 and Eca109 cells (P < 0.05). Elevated PDGF-D level was closely associated with TNM staging, tumor differentiation and lymph node metastasis (P < 0.05), but not related to the patients' age and gender (P>> 0.05). In addition, down-regulation of PDGF-D expression markedly inhibited proliferation, reduced invasion and induced apoptosis in EC1 cells. More importantly, reduced PDGF-D level evoked the down-regulation of p65 and p-IκBα proteins and elevation of IκBα protein of NF-κB pathway, accompanied with the decreases of bcl-2 and MMP-9 protein expressions and increases of bax protein level and caspase-3 activities. Correctively, our data suggest that PDGF-D plays pivotal roles in the development and progression of ESCC, and combinations with PDGF-D and NF-κB pathway may be effective and feasible molecular targets for therapy of ESCC.

OBJECTIVE

Loss or mutation of the phosphate and tensin homologue (PTEN) is a common genetic abnormality in prostate cancer (PCa) and induces platelet-derived growth factor D (PDGF D) signaling. We examined the role of the PTEN/PDGF axis on radioresponse using a murine PTEN null prostate epithelial cell model.

METHODS

PTEN wild-type (PTEN+/+) and PTEN knockout (PTEN-/-) murine prostate epithelial cell lines were used to examine the relationship between the PTEN status and radiosensitivity and also to modulate the PDGF D expression levels. PTEN-/- cells were transduced with a small hairpin RNA (shRNA) lentiviral vector containing either scrambled nucleotides (SCRM) or sequences targeted to PDGF D (shPDGF D). Tumorigenesis and morphogenesis of these cell lines were evaluated in vivo via subcutaneous injection of male nude mice and in vitro using Matrigel 3-dimensional (3D) culture. Effects of irradiation on clonogenic survival, cell migration, and invasion were measured with respect to the PTEN status and the PDGF D expression level. In addition, apoptosis and cell cycle redistribution were examined as potential mechanisms for differences seen.

RESULTS

PTEN-/- cells were highly tumorigenic in animals and effectively formed foci in 3D culture. Importantly, loss of PDGF D in these cell lines drastically diminished these phenotypes. Furthermore, PTEN-/- cells demonstrated increased clonogenic survival in vitro compared to PTEN+/+, and attenuation of PDGF D significantly reversed this radioresistant phenotype. PTEN-/- cells displayed greater migratory and invasive potential at baseline as well as after irradiation. Both the basal and radiation-induced migratory and invasive phenotypes in PTEN-/- cells required PDGF D expression. Interestingly, these differences were independent of apoptosis and cell cycle redistribution, as they showed no significant difference.

CONCLUSIONS

We propose that PDGF D represents a potentially promising target for PCa treatment resistance in the absence of PTEN function, and warrants further laboratory evaluation and clinical study.

The molecular mechanisms of the exaggerated growth of vascular smooth muscle cells (VSMC) in hypertension are reviewed based on our previous experimental data. Spontaneously hypertensive rats (SHR)-derived VSMC increasingly express angiotensinogen, cathepsin D and angiotensin-converting enzyme (ACE) mRNAs, compared to cells from normotensive Wistar-Kyoto (WKY) rats, indicating the presence of an Ang II generating system in a homogeneous culture of VSMC from SHR. The produced Ang II then induces TGF-beta. SHR-derived VSMC show the distinct expression and abnormal regulation by Ang II of TGF-beta receptors when compared with cells from WKY rats, which express TGF-beta type II receptor predominantly to induce PDGF A-chain stimulation of VSMC growth. These findings imply that the increased growth of VSMC in hypertension is a primary event independent of high blood pressure, and is associated with endogenous Ang II-related growth factors.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing β-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100β/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

We investigated the effects of hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) on the expression of alpha2- and beta2-adrenergic responses in primary cultures of adult rat hepatocytes. HGF (1 and 5 ng/ml) rapidly stimulated the expression of beta2-adrenergic responses and significantly increased alpha2-adrenergic responses with a maximal response at 21 h. The stimulatory effects of HGF were reduced dependent on the initial plating density and were completely blocked by cycloheximide (5 microM). On the other hand, PDGF (10 ng/ml) significantly increased the beta2-adrenergic response, but only slightly increased the alpha2-adrenergic responses. Expression of the alpha2- and beta2-adrenergic responses by PDGF was independent of the initial plating density. The expression of these responses was blocked by cycloheximide (5 microM). Northern blot analysis of the hepatocyte mRNA showed increased expression induced by the HGF and PDGF of the both alpha2- and beta2-adrenergic receptor mRNA, and this expression was inhibited by actinomycin D (5 ng/ml). These results indicate that the expression of alpha2- and beta2-adrenergic responses produced by these two growth factors is regulated differently by the cell density of the primary cultures. The results suggest that the expression of alpha2- and beta2-adrenergic responses is mediated through increased synthesis of these receptor proteins.

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.

Low molecular weight chitosan (LMWC) (a mixture of chitooligosaccharides with high degrees of polymerization; an average degree of polymerization is 6.8) stimulated mitogenic response to platelet-derived growth factor (PDGF) in a dose-dependent manner in cultured rat vascular smooth muscle cells, and a maximum effect was observed at 100 micrograms/ml. However, the mitogenic response was not induced when cells were incubated with LMWC alone. This stimulatory effect of LMWC on the mitogenic response to PDGF (a competence factor) appeared to resemble the effect of insulin as a progression factor. Chitooligosaccharides with higher degrees of polymerization were more effective, but D-glucosamine, chitobiose, and chitotriose were barely active. LMWC as well as PDGF induced protein tyrosine phosphorylation in vascular smooth muscle cells.

Activation of phosphatidylinositol (PI) 3-kinase, protein kinase A (PKA) and protein kinase C (PKC) is associated with the survival effect elicited by PDGF-AB and TGF-beta1 against the apoptotic inducer 2-deoxy-D-ribose (dRib) in the fat body cell line, IPLB-LdFB, from the insect Lymantria dispar. dRib induces apoptosis and provokes mitochondrial membrane depolarization (MMD). The antioxidant N -acetyl-L-cysteine annuls only the first effect. These findings suggest that apoptosis and MMD are provoked by two different mechanisms, and that dRib induces apoptosis by oxidative stress.

1. Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine DDDDPDGF)-BB-mediated VSMC migration, proliferation and hypertrophy were investigated. 2. We observed that cell stimulated by 5 ng/mL PDGF-BB showed increased migration, proliferation and hypertrophy. These effects were prevented by co-incubation with dopamine, SKF 38393 or YM 435 at 1-10 mumol/L and this prevention was reversed by Sch 23390 (1-10 mumol/L), a specific DDD (PLD), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activity was significantly suppressed by co-incubation with dopamine. 3. These results suggest that vascular DD, PKC and MAPK activity.

Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine DDPDGF) BB-mediated VSMC migration, proliferation, and hypertrophy were studied. We observed that cells stimulated by 5 ng/ml PDGF BB showed increased migration, proliferation and hypertrophy. These effects were prevented by coincubation with dopamine, SKF 38,393, or YM 435 at 1-10 mumol/l, and this prevention was reversed by Sch 23,390 (1-10 mumol/l), a specific DPDGF-BB (5 ng/ml)-mediated activation of phospholipase D (PLD), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular DD, PKC and MAPK activity.

The aim of our study was to investigate the influence of angiotensin-converting enzyme (ACE) inhibition and angiotensin II receptor blockage on the renal function by light microscopic and immunohistochemical findings in a rat model of tacrolimus nephrotoxicity. Thirty-two male Wistar rats were divided into four groups of eight: G1 = control group; G2-G3, G4 = Tacrolimus (Tac) 1 mg/kg/d intraperitoneally (ip); G3 (Tac + Q) = ip Tac and peroral quinapril 10 mg/kg; and G4 (Tac + V) = Tac and valsartan 40 mg/d. Serum blood urea nitrogen (BUN), creatinine, and creatinine clearance were measured before and at the end of the study period. Renal tissues were assessed for light microscopic findings of tacrolimus toxicity. Transforming growth factor-beta, VEGF, PDGF, BMP-7, and interleukin-6 (IL-6) expression were semiquantitatively scored after immunohistochemical staining. At the end of the study period serum BUN and creatinine levels were increased in all groups, but creatinine clearance was not significantly changed between the groups. Afferent arteriolopathy was significantly less pronounced in G3 versus G2 and G4. Interstial fibrosis was significantly less pronounced in G3 and G4 versus G2. TGF-beta, PDGF, and IL-6 expression were significantly increased in G2, G3, and G4 compared to G1, and in G2 compared to G3 and G4. BMP-7 expression was significantly decreased in G2, G3, and G4 compared to G1, whereas the differences between G2, G3, and G4 failed to reach statistical significance. In conclusion, the results of our study suggested that renin angiotensin inhibition down-regulates fibrogenic cytokine expression in rats displaying tacrolimus nephrotoxicity.