Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.

Herbal hepatotoxicity has been increasingly reported in clinical context, but the underlying mechanisms are poorly understood. Saikosaponin D (SSD) is a major component of saikosaponins isolated from Bupleurum falactum, a herb that has been linked to hepatotoxicity. Our current study was to examine the toxic effect of SSD on human hepatocyte LO2 cells and explore the possible mechanism. The results demonstrated that SSD reduced cell viability and led to dramatic morphological alterations in LO2 cells. Hoechst staining and flow cytometry analyses showed that SSD stimulated hepatocyte apoptosis. SSD-treated cells exhibited apparent nuclear condensation and fragmentation, and the apoptotic cells were increased by SSD dose-dependently. Subsequent experiments showed that SSD decreased mitochondrial membrane potential and downregulated Bcl-2 but upregulated Bax. Moreover, caspase-9 and caspase-3 were activated in SSD-treated LO2 cells. These data consistently indicated that SSD stimulated mitochondrial apoptosis in hepatocytes. Mechanistic investigations showed that SSD disrupted p38 signaling and that p38 specific inhibitor SB203580 mimicked the pro-apoptotic effect of SSD. In addition, platelet-derived growth factor-β receptor (PDGF-βR) blocker imatinib reduced p38 phosphorylation and also mimicked the pro-apoptotic effect of SSD in LO2 cells. These data collectively indicated that SSD induced apoptosis by interrupting PDGF-βR/p38 pathway in LO2 hepatocytes.

OBJECTIVE

To evaluate the therapeutic effect of Chunggan extract (CGX), a modified traditional Chinese hepatotherapeutic herbal, on the dimethylnitrosamine (DMN)-induced chronic liver injury model in rats.

METHODS

Liver injuries were induced in Wistar rats by injection of DMN (ip, 10 mg/mL per kg) for 3 consecutive days per week for 4 wk. The rats were administered with CGX (po, 100 or 200 mg/kg per day) or distilled water as a control daily for 4 wk starting from the 15(th) d of the DMN treatment. Biochemical parameters (serum albumin, bilirubin, ALP, AST and ALT), lipid peroxides, hydroxyproline, as well as histological changes in liver tissues were analyzed. In addition, gene expression of TNF-alpha, TGF-beta, TIMP-1, TIMP-2, PDGF-beta, and MMP-2, all of which are known to be associated with liver fibrosis, were analyzed using real-time PCR.

RESULTS

CGX administration restored the spleen weight to normal after having been increased by DMN treatment. Biochemical analysis of the serum demonstrated that CGX significantly decreased the serum level of ALP (P < 0.05), ALT (P < 0.01), and AST (P < 0.01) that had been elevated by DMN treatment. CGX administration moderately lowered lipid peroxide production and markedly lowered hydroxyproline generation caused by DMN treatment in accordance with histopathological examination. DMN treatment induced a highly up-regulated expression of TNF-alpha, TGF-beta, TIMP-1, TIMP-2, PDGF-beta, and MMP-2. Of these, the gene expression encoding PDGF-beta and MMP-2 was still further enhanced 2 wk after secession of the 4-wk DMN treatment, and was remarkably ameliorated by CGX administration.

CONCLUSIONS

CGX exhibits hepatotherapeutic proper-ties against chronic hepatocellular destruction and consequential liver fibrosis.

In human coronary artery vascular smooth muscle (hcaVSM) cells, the mechanisms that mediate the antiproliferative effects of ligands for the peroxisome proliferator-activated receptor-gamma (PPAR gamma) and the retinoid X receptor-alpha (RXR alpha) are unclear. Dimerization of PPAR gamma with RXR alpha and occupancy by both ligands is required for maximal activation. Accordingly, we determined whether the antiproliferative activity of the PPAR gamma ligands, troglitazone or 15-deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2), was enhanced with the RXR alpha ligand, 9-cis-retinoic acid (9-cis-RA). Incubation of actively proliferating hcaVSM cells with either troglitazone or 15d-PGJ2 resulted in a dose-dependent inhibition of proliferation with half-maximal inhibitory concentrations (IC(50)s) of 13 and 2 microM, respectively. Quiescent cells incubated with troglitazone or 15d-PGJ2 and subsequently stimulated with PDGF-BB showed a concentration-dependent decrease in the active form of MAP kinase, suggesting that inhibition of cell growth by troglitazone may involve the MAP kinase pathway, an important growth activation pathway in VSM cells. Incubation of cells with either 0.1 or 1.0 microM 9-cis-RA inhibited cell growth to a similar degree. Addition of troglitazone or 15d-PGJ2 to cells in combination with either concentration of 9-cis-RA resulted in a striking increase in growth inhibition, and was accompanied by an approximately 4-fold reduction in the IC(50)s for both PPAR gamma ligands. These findings imply that RXR alpha activation by 9-cis-RA synergistically enhanced inhibition of hcaVSM cell growth. The precise nature of this cooperative interaction between PPAR gamma and RXR alpha remains to be determined.

Stimulation of quiescent Swiss 3T3 cells with platelet-derived growth factor (PDGF) increased the initial rate of cytosolic phospholipase A2 activity by 95 +/- 6% over extracts from control cells. Cytosolic phospholipase A2 activity increased rapidly following PDGF treatment (near maximum stimulation by 2.5 min) and was dose-dependent (EC50 = 2 ng/ml). Epidermal growth factor, vasopressin, and phorbol 12,13-dibutyrate also increased cytosolic phospholipase A2 activity but did not produce a sustained mobilization of arachidonic acid in these cells. Detailed kinetic analysis of PDGF-induced arachidonic acid mobilization revealed a biphasic release of 3H radioactivity into the extracellular medium. A first, rapid phase, occurred within 15 min which, like the activation of cytosolic phospholipase A2 activity, was independent of de novo RNA and protein synthesis. After 20 min of stimulation, a second phase became evident which accounts for the majority of arachidonic acid mobilized by PDGF. This second phase was abolished in the presence of either cycloheximide or actinomycin D. Both inhibitors blocked the release of arachidonic acid rather than inhibiting cyclooxygenase activity and consequently prostaglandin E2 production. These findings demonstrate a biphasic mobilization of arachidonic acid in Swiss 3T3 cells by PDGF. Cytosolic phospholipase A2 activity could contribute to the rapid first phase but not the second major phase, which is dependent upon de novo protein synthesis.

Platelet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound the BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP3). Neither pertussis toxin nor short-term (10 min) treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E2. Furthermore, mesangial cells secreted PDGF-AA into the culture supernatant.

Platelet derived growth factor is involved in the autocrine growth stimulation of malignant cells, the stimulation of angiogenesis and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the fibrosarcoma cell microenvironment. Aim of the present study was to examine the effects of glycosaminoglycans on the mitogenic function of platelet derived growth factor in two human fibrosarcoma cell lines (B6FS, HT1080). For this purpose exogenously added glycosaminoglycans, regulators of endogenous glycosaminoglycan synthesis (sodium chlorate as selective inhibitor and beta-D-xyloside as a stimulator) and specific glycosidases to cleave cell-associated glycosaminoglycans, were utilized. Platelet derived growth factor demonstrated a growth stimulating effect on B6FS, whereas no effect was evident on HT1080 fibrosarcoma cells. Beta-D-xyloside had no effect on the basal level or the platelet derived growth factor-induced cell proliferation, whereas sodium chlorate severely reduced the basal level of proliferation in both cell lines. Significant co-stimulatory effects of chondroitin sulfate A in combination with platelet derived growth factor BB on the growth of HT1080 and B6FS cells were found. The co-stimulatory effect of chondroitin sulfate A was not due to transcriptional up regulation of platelet derived growth factor receptors genes, but rather to more efficient signalling of tyrosine kinase receptors. In conclusion, this study shows that chondroitin sulfate A can enhance the mitogenic activity of platelet-derived growth factor in fibrosarcoma cells utilizing a pathway which involves tyrosine kinases. This result introduces a new modulating role for chondroitin sulfate in signalling pathways critical for cancer growth.

BACKGROUND

Increased immunoreactivity of platelet-derived growth factor (PDGF)-AA, -Ralpha, and -Rbeta in intimal cells correlates with the development of cardiac allograft arteriosclerosis, a condition for which there is little or no current therapy. Therefore, we hypothesized that PDGF may have a rate-limiting role in the development of this disease.

RESULTS

The hypothesis was tested in a rat model of heterotopic cardiac and aortic allografts using dark agouti (AG-B4, RT1(a)) donors and Wistar-Furth (AG-B2, RT1(u)) recipients. The recipients received CGP 53716, a selective PDGF-R protein tyrosine kinase inhibitor, 50 mg. kg-1. d-1, or vehicle for 60 days. Cardiac allograft recipients also received background cyclosporin A immunosuppression. Our results demonstrate that CGP 53716 significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac and aortic allograft recipients. When rat coronary smooth muscle cells were stimulated in vitro with PDGF-AA or -BB in the presence of interleukin-1beta or tumor necrosis factor-alpha, CGP 53716 significantly inhibited only AA-ligand-induced but not BB-ligand-induced replication. Concomitantly, in quantitative reverse transcriptase-polymerase chain reaction, interleukin-1beta or tumor necrosis factor-alpha stimulation specifically upregulated the expression of PDGF-Ralpha mRNA but not of other ligand or receptor genes in cultured smooth muscle cells.

CONCLUSIONS

We conclude that a PDGF-AA/Ralpha-dependent cycle is induced in the generation of allograft arteriosclerosis that may be inhibited by blocking of signaling downstream of PDGF-R.

In this study, we explored the complex interactions between platelet-derived growth factor (PDGF) and N-methyl-d-aspartate receptor (NMDAR) and their effect on the excessive proliferation and migration of smooth muscle cells leading to obstructed arteries in pulmonary arterial hypertension (PAH). We report lower expression of glutamate receptor NMDA-type subunit 2B (GluN2B), a subunit composing NMDARs expected to affect cell survival/proliferation of pulmonary artery smooth muscle cells (PASMCs), in PAH patient lungs. PASMC exposure to PDGF-BB stimulated immediate increased levels of phosphorylated Src family kinases (SFKs) together with increased phosphorylated GluN2B (its active form) and cell surface relocalization, suggesting a cross talk between PDGFR-recruited SFKs and NMDAR. Selective inhibition of PDGFR-β or SFKs with imatinib or A-419259, respectively, on one hand, or with specific small-interfering RNAs (siRNAs) on the other hand, aborted PDGF-induced phosphorylation of GluN2B, thus validating the pathway. Selective inhibition of GluN2B using Rö25-6981 and silencing with specific siRNA, in the presence of PDGF-BB, significantly increased both migration and proliferation of PASMCs, thus strengthening the functional importance of the pathway. Together, these results indicate that GluN2B-type NMDAR activation may confer to PASMCs antiproliferative and antimigratory properties. The decreased levels of GluN2B observed in PAH pulmonary arteries could mediate the excessive proliferation of PASMCs, thus contributing to medial hyperplasia and PAH development.

Oxidant/antioxidant imbalance is thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Therefore, antioxidants, such as superoxide dismutase (SOD), are expected to have an inhibitory potential against IPF. To elucidate whether a lecithinized SOD (phosphatidylcholine [PC]-SOD) has the potential to be a new therapeutic agent for IPF, we investigated the inhibitory effects of PC-SOD at doses of 1 mg/kg/d (low dose) and 10 mg/kg/d (high dose) and of methylprednisolone (mPSL) on bleomycin (BLM)-induced pulmonary fibrosis in mice. Histopathologic evaluation and lung hydroxyproline content revealed that the severity of fibrosis was attenuated in mice treated with low-dose PC-SOD, whereas no significant effect was observed in other mice. In bronchoalveolar lavage fluid on Day 1 after treatment with BLM, BLM-induced increases in total cell number, populations of lymphocytes and neutrophils, and expression of messenger RNA for interleukin-1beta and platelet-derived growth factor (PDGF)-A were significantly suppressed in PC-SOD-treated mice. The suppression of PDGF-A expression was significantly greater in mice treated with low-dose PC-SOD than in mice treated with high-dose PC-SOD or mPSL. In summary, this study demonstrated the inhibitory effects of low-dose PC-SOD on the development of pulmonary fibrosis, which indicates the potential usefulness of PC-SOD as a new treatment agent for IPF or at least for BLM-induced pulmonary fibrosis in humans.

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.

Cigarette smoking is implicated in the formation of occlusive vascular diseases. Nicotine's role in this process is incompletely understood. Nicotine's effect on human aortic vascular smooth muscle cells (HaVSMC) and the role of the nicotinic receptor (nAChR), platelet-derived growth factor (PDGF), and the PDGF-receptor (PDGF-R) in this response were studied. Nicotine's mitogenic effect was characterized by three methods: thymidine incorporation, a viability/proliferation assay based on metabolic conversion of tetrazolium salt to formazan dye and cell counting. Nicotine administration (10(-6) M) stimulated cell cycle entry marked by increased DNA synthesis, PCNA and cyclin D1 production, and increased cell division. Nicotinic receptor blockade with d-tubocurarine, a nicotinic AchR blocker, decreased nicotine-induced DNA synthesis, and cell division (0.33 +/- 0.04, 0.77 +/- 0.31-fold decrease, respectively). Nicotine increased cellular PDGF-BB transcript levels and protein release (ELISA: 1.6 +/- 0.5-fold increase) but not PDGF-AA or PDGF-AB release. Nicotine increased PDGFbeta-receptor protein content. PDGF inactivation with anti-PDGF antibody abolished nicotine-induced DNA synthesis (1.9 +/- 0.08-fold decrease). PDGF-R blockade with the PDGF-R antagonist tyrphostin AG 1295 decreased nicotine-induced DNA synthesis and cell division (0.25 +/- 0.01, 0.44 +/- 0.2-fold decrease, respectively). PDGF-R blockade reversed nicotine-stimulated increases in PDGF release, PDGF-BB transcripts, and PDGF-receptor levels (0.68 +/- 0.34; 0.46 +/- 0.01; 0.28 +/- 0.01-fold decrease, respectively). In conclusion, nicotine-mediated activation of nAChRs increases PDGF-BB transcription and protein production as well as PDGF beta-receptor levels. PDGF-BB/PDGF-R interaction is vital in nicotine's mitogenic actions on human aortic smooth muscle cells.

Cells and tissues have developed a variety of ways of responding to a hostile environment, be it from drugs (toxins) or radiation (summarized in Fig. 1). Three categories of radiation damage limitation are: (i) DNA repair (ii) changes in cellular metabolism (iii) changes in cell interaction (cell contact or tissue-based resistance; whole organism based resistance). DNA repair has been evaluated predominantly by the study of repair-deficient mutants. The function of the repair genes they lack is not fully understood, but some of their important interactions are now characterized. For example, the interaction of transcription factors with nucleotide excision repair is made clear by the genetic syndromes of xeroderma-pigmentosum groups B, D and G. These diseases demonstrate ultraviolet light sensitivity and general impairment of transcription: they are linked by impaired unwinding of the DNA required for both transcription and repair. The transfer of DNA into cells is sometimes accompanied by a change in sensitivity to radiation, and this is of special interest when this is the same genetic change seen in tumors. DNA repair has a close relationship with the cell cycle and cell cycle arrest in response to damage may determine sensitivity to that damage. DNA repair mechanisms in response to a variety of drugs and types of radiation can be difficult to study because of the inability to target the damage to defined sequences in vivo and the lack of a satisfactory substrate for in vitro studies. Changes in cellular metabolism as a result of ionizing radiation can impart radiation resistance, which is usually transient in vitro, but may be more significant in vivo for tissues or tumors. The mechanisms by which damage is sensed by cells is unknown. The detection of free radicals is thought likely, but distortion to DNA structure or strand breakage and a direct effect on membranes are other possibilities for which there is evidence. Changes in extracellular ATP occur in response to damage, and this could be a direct membrane effect. External purinergic receptors can then be involved in signal transduction pathways resulting in altered levels of thiol protection or triggering apoptosis. Changes in the functional level of proteins as a consequence of ionizing radiation include transcription factors, for example c-jun and c-fos; cell cycle arrest proteins such as GADD (growth arrest and DNA damage inducible proteins) and p53; growth factors such as FGF, PDGF; and other proteins leading to radioresistance.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

To investigate and compare the inhibitory effects of rapamycin in the different stages of liver fibrosis.

METHODS

We performed bile duct ligation (BDL) in male Wistar rats (n = 24). The experimental rats were classified into four groups: the BDL(+)/Rapa(-) group (un-treated control, n = 4), the BDL(+)/Rapa(+) group (treated 14 d after BDL, n = 8), the BDL(+)/Rapa(++) group (treated on the day after BDL, n = 8), and the BDL(-)/Rapa(-) group (un-treated, sham -operated control, n = 4). The BDL(+)/Rapa(+) and BDL(+)/Rapa(++) groups were administered rapamycin (2 mg/kg) for 28 d. The liver tissues were tested by immunohistochemical staining for α-smooth muscle actin (α-SMA) and cytokeratin.

RESULTS

The liver mRNA levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF) were measured using the polymerase chain reaction. The protein levels of liver p70s6K and p-p70s6k were determined using Western blotting. α-SMA expression was lowest in the BDL(+)/Rapa(++)group. TGF-β1 and PDGF expression levels in the rapamycin-treated group were lower than those in the un-treated group and higher than those in the control groups (TGF-β1: 0.23 ± 0.00 vs 0.34 ± 0.01, 0.23 ± 0.0 vs 0.09 ± 0.00, P < 0.0001; PDGF: 0.21 ± 0.00 vs 0.34 ± 0.01, 0.21 ± 0.0 vs 0.09 ± 0.00, P < 0.0001). The p70s6k and p-p70s6k levels decreased in the treated groups and were lowest in the BDL(+)/Rapa(++)group (p70s6k: 1.05 ± 0.17 vs 1.30 ± 0.56, 0.40 ± 0.01 vs 1.30 ± 0.56, P < 0.0001; p-p70s6k: 1.40 ± 0.5 vs 1.67 ± 0.12, 0.70 ± 0.01 vs 1.67 ± 0.12, P < 0.0001).

CONCLUSIONS

The results of our study indicate that rapamycin has inhibitory effects on liver fibrosis, and the treatment is most effective in the early stages of fibrosis.

BACKGROUND

The aim was to assess wound healing when parenteral fish-oil emulsion is given to rats receiving dexamethasone.

METHODS

For 5 days after skin wounding, group S (control; n = 7) received saline 1 mL/kg intraperitoneal (IP); group D (n = 7), dexamethasone 0.2 mg/kg IP; and group DO (n = 9), dexamethasone 0.2 mg/kg IP plus 1 mL/kg Omegaven (Fresenius Kabi, Austria). Wound specimens were assessed for hydroxyproline level, wound depth, histology (epidermal/dermal regeneration, granulation tissue thickness, and angiogenesis), and expression of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor-AA (PDGF-AA).

RESULTS

Compared with D and DO specimens, controls had higher hydroxyproline (p < .01), deeper wounds (p < .05), and better histologic scores (p < .01 angiogenesis; others p < .05). There were no significant differences between the group D and DO means for hydroxyproline level, wound depth, or histologic scores (p>> .05 for all). Controls had higher TGF-beta expression scores than the other groups (p < .01 for both) and a higher PDGF-AA expression score than group DO (p < .01). Groups D and DO had statistically similar TGF-beta scores, but group D had a higher PDGF-AA score (2.71 +/- 0.75 vs 1.55 +/- 0.72, respectively; p < .05).

CONCLUSIONS

According to the parameters we studied, adding parenteral omega-3 and omega-6 fatty acids to the nutrition regimen of rats treated with dexamethasone does not seem to have adverse effects on wound healing, and effects on wound healing may not need to be considered when determining if these agents should be supplemented in nutrition support regimens.

In this study, we found that wounding of a confluent monolayer of squamous cell carcinoma (SCC) cells induced epithelial-mesenchymal transition (EMT) specifically at the edge of the wound. This process required the combined stimulation of TGFβ, TNFα, and PDGF-D. Such a combined cytokine treatment of confluent monolayers of the cells upregulated the expression levels of Snail and Slug via PI3K. The PI3K downstream effector, AKT, was dispensable for the upregulation of Snail and Slug, but essential for enabling EMT in response to upregulation of Snail and Slug.

We previously reported that fibroblasts migrating within 3-D collagen matrices move independently, whereas fibroblasts within 3-D fibrin matrices form an interconnected network. Similar networks have been identified previously during in vivo corneal wound healing. In this study, we investigate the role of fibronectin in mediating this mechanism of collective cell spreading, migration and patterning.

To assess cell spreading, corneal fibroblasts were plated within fibrillar collagen or fibrin matrices. To assess migration, compacted cell-populated collagen matrices were nested inside cell-free fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without RGD peptide, anti-α5 or anti-fibronectin blocking antibodies. In some experiments, LifeAct and fluorescent fibronectin were used to allow dynamic assessment of cell-induced fibronectin reorganization. 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, F-actin, α5 integrin and fibronectin organization.

Corneal fibroblasts within 3-D fibrin matrices formed an interconnected network that was lined with cell-secreted fibronectin. Live cell imaging demonstrated that fibronectin tracks were formed at the leading edge of spreading and migrating cells. Furthermore, fibroblasts preferentially migrated through fibronectin tracks laid down by other cells. Interfering with cell-fibronectin binding with RGD, anti α5 integrin or anti fibronectin antibodies inhibited cell spreading and migration through fibrin, but did not affect cell behavior in collagen.

In this study, a novel mode of cell patterning was identified in which corneal fibroblasts secrete and attach to fibronectin via α5β1 integrin to facilitate spreading and migration within 3-D fibrin matrices, resulting in the formation of localized fibronectin tracks. Other cells use these fibronectin tracks as conduits, resulting in an interconnected cell-fibronectin network.

Platelet-derived growth factor (PDGF) is one of the growth factors that reportedly regulates cell growth and division of mesenchymal cells. Although PDGF isoforms and their receptors reportedly play a pivotal role in mesenchymal stem cell regulation, there is a paucity of literature reviewing the role of PDGF in adipose-derived stem cells (ASCs). Therefore, we summarized previous reports on the expression and functional roles of PDGF and its receptor isoforms in this review. In addition, we examined findings pertaining to underlying molecular mechanisms and signaling pathways with special focus on PDGF-D/PDGFRβ. ASCs only express PDGF-A, -C, -D, PDGFRα, and PDGFRβ. PDGFRα expression decreases with adipocyte lineage, while PDGFRβ inhibits white adipocyte differentiation. In addition, PDGFRβ induces proliferation, migration, and angiogenesis and up-regulates the expression of paracrine factors in ASCs. Although PDGF-B and -D mediate their functions mainly by PDGFRβ and ROS generation, there are many differences between them in terms of regulating ASCs. PDGF-D is endogenous, generates ROS via the mitochondrial electron transport system, and regulates the autocrine loop of ASCs in vivo. Furthermore, PDGF-D has stronger mitogenic effects than PDGF-B.

Adipose derived stem cells (ASC) differentiate into a Schwann cell (SC)-like phenotype but the signalling pathways mediating this are unknown. We hypothesised that notch might be involved, given its important role in regulating SC development. Rat ASC were differentiated using bFGF, PDGF, GGF-2 and forskolin. RT-PCR analysis showed that mRNA for notch-1 and notch-2 receptors and the notch responsive gene, hes-1, were expressed throughout the differentiation process whereas jagged-1 a notch ligand, and the hey-1 gene were markedly down-regulated. In contrast delta-1 was up-regulated with differentiation and was strongly expressed by rat primary SC. Treatment of ASC with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor which blocks notch signalling, had no effect on up-regulation of SC proteins S100 or GFAP during differentiation. Furthermore, when co-cultured with NG108-15 neurons, differentiated ASC cultures treated in the absence or presence of DAPT enhanced neurite outgrowth to similar levels. Differentiated ASC expressed PMP-22 but P0 was only present when co-cultured with dorsal root ganglia neurons. DAPT did not affect the expression of these myelin proteins. Thus, ASC express components of the notch signalling pathway but our studies suggest notch is unlikely to play a role in the neurotrophic activity and myelination capability of ASC differentiated into SC-like cells.

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

Hepatocyte growth factor (HGF), a cytokine which is generally produced by mesenchymal cells, has mitogenic, motogenic and morphogenic activities in epithelial cells and it also has tumor-suppressing activities. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. We examined the effects of ascorbic acid (AsA), which stimulates the proliferation of fibroblasts, and its stable derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), on HGF production by human skin fibroblasts. Basal HGF secretion was significantly stimulated by more than 0.1 mM AsA or AA-2G. Both vitamins synergistically enhanced HGF secretion stimulated by growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), cholera toxin and other inducers. Induction by EGF or bFGF was most markedly potentiated by the vitamins. HGF production by the KG-1 human leukemia cell line was also augmented by AsA or AA-2G. Another stable AsA derivative, ascorbic acid 2-phosphate (AA-2P) effectively promoted basal and EGF-induced HGF secretion by the fibroblasts, but ascorbic acid 2-sulfate (AA-2S) was much less effective. Intracellular AsA levels increased after the addition of AA-2G and AA-2P as well as AsA, but not after AA-2S. The effect of AA-2G was completely abrogated by the simultaneous addition of castanospermine, an alpha-glucosidase inhibitor, suggesting that the active form of AA-2G is AsA. Constitutive and EGF-induced HGF gene expression was also up-regulated after adding AsA or AA-2G to the cells. These results indicated that AsA acts alone or in synergy with several inducers to stimulate the production and gene expression of HGF in human skin fibroblasts and that the stable AsA derivative AA-2G is as effective as AsA in promoting HGF production.

Drug-induced liver injury can lead to acute liver failure. Saikosaponin D (SSD) is a major component isolated from the medicinal herb Bupleurum falcatum, which has been linked to hepatotoxicity. We previously reported that SSD disrupted PDGF-βR pathway leading to mitochondrial apoptosis in human LO2 hepatocytes. The present study was aimed at further exploring the underlying mechanisms in vitro and in vivo. We initially determined the concentration range of SSD at up to 2μM for subsequent apoptosis examinations. SSD significantly upregulated Fas expression, promoted caspase-8 cleavage and activated the pro-apoptotic protein Bid in LO2 cells. Moreover, SSD reduced the abundance of cytochrome c in mitochondria and increased the cleaved-caspase-3 in LO2 cells, but did not apparently affect PI3K/AKT, ERK and STAT3 pathways that are involved in cell fate regulation. Experiments in vivo showed that one-week treatment with SSD at 300 mg/kg significantly elevated the liver/body weight ratio and caused histological injury in mouse liver. Furthermore, SSD treatment induced massive hepatocyte apoptosis, and significantly downregulated Bcl-2 but upregulated Bax in mouse liver. Taken together, these results revealed a specific mechanism of activation of extrinsic apoptosis pathway and Bid by SSD, which was involved in SSD-induced mitochondrial apoptosis in hepatocytes and potential hepatotoxicity.

Dexamethasone-releasing PLGA poly(lactic-co-glycolic acid) microsphere/PVA (polyvinyl alcohol) hydrogel composite coatings have been shown to prevent the foreign body reaction (FBR) to subcutaneous implants in small and large animal models. Such coatings were developed to extend the lifetime of implantable biosensors. However, long-term exposure of tissue to low levels of dexamethasone results in a reduction in blood vessel density due to the anti-angiogenic effect of dexamethasone. This mild effect, while not threatening to the subject's health, may interfere with analyte detection and the sensor response time over the long-term. The present work is focused on the development of coatings that deliver combinations of three tissue response modifiers (TRMs): dexamethasone, VEGF (vascular endothelial growth factor) and PDGF (platelet derived growth factor). Dexamethasone, VEGF and PDGF prevent the FBR, increase angiogenesis and promote blood vessel maturation (which increases blood flow), respectively. To minimize any potential interference among these three TRMs (for example, PDGF increases fibrosis), the relative doses of dexamethasone, VEGF and PDGF were adjusted. It was determined that: a) all three TRMs are required for maximum promotion of angiogenesis, blood vessel maturation and prevention of the FBR; b) VEGF has to be administered at higher doses than PDGF; c) an increase in dexamethasone dosing must be accompanied by a proportional increase in growth factor dosing; and d) modification of the TRM ratio can achieve a constant capillary density throughout the implantation period which is important for applications such as biosensors to maintain sensitivity and a stable sensor baseline. Moreover, an osmosis-driven process for encapsulation of proteins in PLGA microspheres that showed low burst release was developed.

As the aged population continues to markedly increase worldwide, the incidences of diabetes mellitus (DM) and cardiovascular disease (CVD) are increasing. In this study, we investigated the effects of aging, DM, and antiplatelet drugs on growth factors and anti-aging proteins in platelet-rich plasma (PRP). The study participants were classified into the following four groups: Group A, healthy individuals aged ≤45 years; Group B, healthy individuals aged >45 years; Group C, DM patients aged >45 years; and Group D, CVD patients aged >45 years taking antiplatelet drugs. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, vascular endothelial growth factor (VEGF)-A, tissue inhibitor of metalloproteinase 2 (TIMP2), insulin-like growth factor 1 (IGF-1), growth differentiation factor (GDF)11, and clusterin in PRP samples were determined to analyze the effects of aging, DM, and antiplatelet drugs. Overall, the concentrations of IGF-1, TIMP2, and clusterin did not vary significantly between the four groups. The concentrations of PDGF-AB/BB (P = 0.010), VEGF-A (P = 0.000), and GDF11 (P = 0.026) were significantly different between Group A and Group B. Further, the concentrations of EGF (P = 0.000) and GDF11 (P = 0.000) were significantly different between Groups B and C. The concentrations of EGF (P = 0.001), VEGF-A (P = 0.000), and GDF11 (P = 0.002) significantly differed between Groups A and C. The concentrations of FGF-2 (P = 0.048), PDGF-AA (P = 0.03), and GDF11 (P = 0.001) were significantly different between Groups B and D. The concentrations of PDGF-AB/BB (P = 0.032), VEGF-A (P = 0.010), and GDF11 (P = 0.02) significantly differed between Groups A and D. We found that PRP contains high concentrations of the growth factors, TIMP2 and GDF11. Aging, DM, and antiplatelet drugs can decrease the concentration of some growth factors and GDF11, which weakens the regenerative capacity and anti-aging effects of PRP and reduces the quality of PRP.