BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder marked by relentless fibrosis and damage of the lung architecture. A growing body of evidence now suggests that IPF progresses as a result of aberrant epithelial-fibroblast crosstalk. Injured epithelia are a major source of growth factors such as PDGF which guide resident fibroblasts to injury sites.

RESULTS

In this study, we utilized a novel co-culture system to investigate the effect of fibroblast phenotype on their response to epithelial injury. Fibroblasts from normal lungs (NHLF) responded to epithelial injury and populated the wound site forming a fibroblast plug/mechanical barrier which prevented epithelial wound closure. IPF fibroblasts were impaired in their response to epithelial injury. They also expressed reduced PDGFRα compared to NHLFs and were defective towards PDGF-AA mediated directional movement. Neutralization of PDGF-AA and pan-PDGF but not PDGF-BB reduced the injury response of NHLFs thereby preventing the formation of the mechanical barrier and promoting epithelial wound closure. Co-culture of epithelial cells with IPF fibroblasts led to marked increase in the levels of pro-fibrotic growth factors - bFGF and PDGF and significant depletion of anti-fibrotic HGF in the culture medium. Furthermore, IPF fibroblasts but not NHLFs induced a transient increase in mesenchymal marker expression in the wound lining epithelial cells. This was accompanied by increased migration and faster wound closure in co-cultures with IPF fibroblasts.

CONCLUSIONS

Our data demonstrate that the IPF fibroblasts have an aberrant repair response to epithelial injury.

BACKGROUND

The leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins constitute an integral membrane protein family that has three members: LRIG1, LRIG2, and LRIG3. LRIG1 negatively regulates growth factor signaling, but little is known regarding the functions of LRIG2 and LRIG3. In oligodendroglial brain tumors, high expression of LRIG2 correlates with poor patient survival. Lrig1 and Lrig3 knockout mice are viable, but there have been no reports on Lrig2-deficient mice to date.

RESULTS

Lrig2-deficient mice were generated by the ablation of Lrig2 exon 12 (Lrig2E12). The Lrig2E12-/- mice showed a transiently reduced growth rate and an increased spontaneous mortality rate; 20-25% of these mice died before 130 days of age, with the majority of the deaths occurring before 50 days. Ntv-a transgenic mice with different Lrig2 genotypes were transduced by intracranial injection with platelet-derived growth factor (PDGF) B-encoding replication-competent avian retrovirus (RCAS)-producing DF-1 cells. All injected Lrig2E12+/+ mice developed Lrig2 expressing oligodendroglial brain tumors of lower grade (82%) or glioblastoma-like tumors of higher grade (18%). Lrig2E12-/- mice, in contrast, only developed lower grade tumors (77%) or had no detectable tumors (23%). Lrig2E12-/- mouse embryonic fibroblasts (MEF) showed altered induction-kinetics of immediate-early genes Fos and Egr2 in response to PDGF-BB stimulation. However, Lrig2E12-/- MEFs showed no changes in Pdgfrα or Pdgfrβ levels or in levels of PDGF-BB-induced phosphorylation of Pdgfrα, Pdgfrβ, Akt, or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Overexpression of LRIG1, but not of LRIG2, downregulated PDGFRα levels in HEK-293T cells.

CONCLUSIONS

The phenotype of Lrig2E12-/- mice showed that Lrig2 was a promoter of PDGFB-induced glioma, and Lrig2 appeared to have important molecular and developmental functions that were distinct from those of Lrig1 and Lrig3.

BACKGROUND

Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model.

METHODS

Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry.

RESULTS

When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates.

CONCLUSIONS

The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.

Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).

Platelet-derived growth factor (PDGF-BB homodimer) and transforming growth factor-beta 1 (TGF-beta 1) are potent wound-healing hormones that accelerate incisional repair. To identify more precisely and quantitatively the stage(s) of wound healing influenced by growth-factor therapy, we investigated the three sequential tissue repair processes--inflammatory cell influx, intracellular procollagen type I (PC-I) synthesis, and collagen cross-linking--in recombinant growth factor--treated wounds. Using newly developed automated, quantitative image-analysis techniques, we observed that PDGF-BB markedly augmented the directed migration of macrophages into wounds during the first week after wounding and triggered and earlier and more sustained influx of PC-I--containing fibroblasts into the wound when compared with results in TGF-beta 1-treated or control wounds (p = 0.015 at day 2; p = 0.007 at day 21). In contrast, automated image analysis revealed TGF-beta 1-treated wound fibroblasts had a nearly twofold increase in intracellular levels of PC-I protein when compared with PDGF-BB-treated or control wound fibroblasts (p = 0.004 at day 4). However, the influence of TGF-beta 1 was transient, and the longer duration of PDGF-BB activity suggested a later influence, perhaps on the collagen remodeling phase, which is ultimately required for increased wound strength. To address this possibility, collagen cross-linking in growth factor-treated wounds was inhibited by beta-aminoproprionitrile (BAPN) treatment, and wound breaking strength was analyzed. Both PDGF-BB and TGF-beta 1 continued to enhance repair in BAPN-treated rats, indicating that they do not function primarily at the level of collagen cross-linking. Thus, PDGF-BB appears to enhance the inflammatory phase of wound healing to indirectly trigger PC-I synthesis, whereas TGF-beta 1 quantitatively enhances PC-I synthesis directly, accounting for their differing duration of activities within healing wounds.

Mechanisms underlying stimulation of platelet-derived growth factor (PDGF) beta-receptors expressed on connective tissue cells in human colorectal adenocarcinoma were investigated in this study. PDGF-AB/BB, but not PDGF receptors, was expressed by tumor cells in situ, as well as in tumor cell isolates of low passage from human colorectal adenocarcinoma. In an experimental co-culture system, conditioned medium from tumor cells only marginally activated PDGF beta-receptors expressed on fibroblasts. In contrast, co-culturing of the two cell types led to a marked PDGF beta-receptor activation. Functional PDGF-AB/BB was found to be associated with heparinase-I-sensitive components on the tumor cell surface. PDGF-AB/BB, isolated from heparinase-I-sensitive cell surface components, induced a marked activation of PDGF beta-receptors. Furthermore, co-culturing tumor cells together with fibroblasts led to a sustained activation of PDGF beta-receptors expressed on fibroblasts. Double immunofluorescence staining of tissue sections from human colorectal adenocarcinoma, combined with computer-aided image analysis, revealed that nonproliferating tumor cells were the predominant cellular source of PDGF-AB/BB in the tumor stroma. In addition, PDGF-AB/BB-expressing tumor cells were found juxtapositioned to microvascular cells expressing activated PDGF beta-receptors. Confocal microscopy revealed a cytoplasmic and cell-membrane-associated expression of PDGF-AB/BB in tumor cells situated in the stroma. In contrast, epithelial cells situated in normal or tumorous acinar structures revealed only a cell-membrane-associated PDGF-AB/BB expression. The is vitro and in situ results demonstrate that tumor cells not only facilitate but also have the ability to modulate connective tissue cell responsiveness to PDGF-AB/BB in a paracrine fashion, through direct cell-cell interactions in human colorectal adenocarcinoma.

OBJECTIVE

The severity of pulmonary dysfunction and subsequent development of chronic lung disease (CLD) in preterm neonates depends on several factors, among them oxygen administration. The aim of this report is to compare the effects of high-frequency, oscillatory ventilation (HFOV) versus synchronized, intermittent, mandatory ventilation (sIMV) on serum cytokine levels (IL-6, IL-8, IL-10, MCP-1, PDGF-BB, VEGF and TGF-beta1) and ventilator indices during the first week of life. Moreover, CLD development and several other outcomes were compared between the two groups.

METHODS

Randomized clinical trial.

METHODS

Third level NICU.

METHODS

40 preterm neonates with a gestational age between 24 and 29 weeks were randomly (20 per group) assigned to one of the two, above-mentioned ventilation strategies within 30 minutes of birth.

RESULTS

At 1, 3 and 5 days, neonates were monitored by means of ventilator indices and levels of seven pro-inflammatory or anti-inflammatory (pro-fibrotic) cytokines in serum. No clinical or biochemical differences were observed at baseline. The neonates assigned to HFOV benefited from early and sustained improvement in gas exchange, with earlier extubation and lower incidence of CLD, as compared to the neonates assigned to sIMV treatment, and showed a significant reduction of serum IL-6, IL-8 and IL-10 over time only when the HFOV treatment was administered. In addition, at days 3 and 5, the IL-6 levels were significantly lower in the HFOV group as compared to sIMV patients.

CONCLUSIONS

The results of this randomized clinical trial support the hypothesis that early use of HFOV, combined with an optimum volume strategy, has a beneficial effect, reducing serum levels of pro-inflammatory cytokines and consequently the acute phase leading to lung injury.

During acute lung injury, there is an outpouring of growth factors into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived growth factor (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, 15, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38-40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38-40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody. The presence of these two peptides varied independently with time after exposure to bleomycin. The 38-40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant growth-promoting activity for fibroblasts present in concentrated ELF during the course of injury.(ABSTRACT TRUNCATED AT 250 WORDS)

We designed a growth factor release system to potentially stabilize neovascularization in the treatment of ischemic tissue. In this study, the release of PDGF-BB and TGF-β1 was controlled with distinct kinetics from injectable PEGylated fibrin gels. Growth factors can be loaded into PEGylated fibrin gels via 3 mechanisms: entrapment, conjugation through a homobifunctional amine reactive PEG linker, and physical affinity with the fibrin matrix. PDGF-BB was entrapped during thrombin-mediated crosslinking leading to a diffusion-controlled release over 2days. TGF-β1 was both conjugated through the PEG linker and bound to the matrix via physical affinity, delaying the release rate of TGF-β1 up to 10days. Further, the release rate was highly correlated to gel degradation rate indicating that TGF-β1 release is degradation-controlled. Therefore, by modulating the molar ratio of PEG to fibrinogen, we were able to control the release rate of TGF-β1 without altering the release kinetics of PDGF-BB. The bioactivity of loaded TGF-β1 was maintained upon release as evidenced by the inhibition of cell proliferation. This system could be expanded to incorporate growth factors loaded via 3 schemes with differing release rates from an injectable system allowing for a high degree of flexibility in other combinational drug delivery and tissue engineering systems.

The objective of the present study was to elucidate the presence and cellular distribution of insulin-like growth factor I (IGF-I), IGF-I receptor (IGF-IR), and IGF binding proteins (IGFBPs) in human uterine tissue at various reproductive stages, and to determine the effect of IGF-I and its interaction with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in endometrial stromal and myometrial smooth muscle cells in primary culture. Using specific antibodies, immunohistochemical observations indicated that luminal and glandular epithelial cells were the major sites of immunoreactive IGF-I, IGF-IR, and IGFBPs 1-4, followed by myometrial smooth muscle and endometrial stromal cells. The immunostaining intensity of IGF-I, IGF-IR, and IGFBPs in endometrial but not myometrial tissue was cycle-dependent and higher in the late proliferative and early/mid-secretory periods than in the late secretory and postmenopausal periods, with little immunostaining at the early proliferative phase of the menstrual cycle. Stromal and smooth cells in primary cell culture also contained immunoreactive IGF-I, IGF-IR, and IGFBPs. IGF-I at 10-100 ng/ml stimulated 3H-thymidine incorporation in quiescent stromal and smooth muscle cells with maximal effect at 100 ng/ml (p < 0.05). However, in the presence of 2% serum, which induces half-maximal stimulation, IGF-I (100 ng/ml) further increased the rate of 3H-thymidine incorporation in stromal but not smooth muscle cells (p < 0.05). The effect of IGF-I was significantly lower than that induced by EGF (10 ng/ml), PDGF-BB (10 ng/ml) and their combination (p < 0.005), and higher in stromal cells from proliferative, than secretory phase of the cycle in the presence of 2% fetal bovine serum, but not serum-free condition (p < 0.005). The effect of IGF-I on myometrial smooth muscle cells was significantly higher than that induced by EGF, but lower than that induced by PDGF-BB or by EGF+PDGF-BB, without the cycle specificity seen with stromal cells. EGF, PDGF-BB, and their combination with IGF-I, but not IGF-I alone, stimulated stromal and smooth muscle cell growth as determined by a cell proliferation assay. The results indicate that human uterine tissue at various reproductive stages contains immunoreactive IGF-I, IGF-IR, and IGFBPs 1-4. Although IGF-I alone was found to be a weak mitogenic factor for stromal and smooth muscle cells, by interacting with EGF and PDGF-BB in a cycle-dependent manner it may regulate the growth and differentiation of these and other uterine cell types.

BACKGROUND

Angiogenesis is a key feature of endometrial development. Inappropriate endometrial vascular development has been associated with recurrent miscarriage (RM) with increased amounts of perivascular smooth muscle cells surrounding them.

METHODS

In the current study, we have used immunohistochemistry to study temporal and spatial expression of a series of angiogenic growth factors (AGFs) and their receptors; vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, platelet-derived growth factor (PDGF)-BB, PDGF-Rα, PDGF-Rβ, transforming growth factor (TGF)-β1, TGF-βRI, TGF-βRII, angiopoietin (Ang)-1, Ang-2 and Tie-2, in the proliferative, early secretory and mid-late secretory phase endometrium from control women as well as in the mid-late secretory phase of women with a history of RM. The AGFs and their receptors studied were immunostained and assessed separately in stromal, vascular smooth muscle, endothelial and glandular epithelial cells. Laser capture microdissection and real-time RT-PCR were used to confirm expression patterns observed by immunohistochemistry.

RESULTS

Most AGFs investigated showed both temporal and spatial expression patterns in normal cycling endometrium. In addition, immunostaining intensity for several AGFs was altered in women with a history of RM, particularly in vascular smooth muscle cells (VSMCs). VSMC expression of TGF-β1, VEGF-R1 and VEGF-R2 was increased while expression of PDGF-BB, TGF-βRI, TGF-βRII, Ang-2, VEGF-A and VEGF-C was reduced.

CONCLUSIONS

This study confirms that the cycling endometrium is a highly angiogenic tissue and that this process is likely to be altered in women with a history of RM and may contribute to the aetiology of this condition.

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF beta receptor (PDGF-betaR) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-betaR downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-betar mRNA expression. Our studies show that pdgf-betar mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-betar mRNA and protein. Suppression of pdgf-betar mRNA in response to Myc is specific, since expression of the related receptor pdgf-alphar is not affected. We further show that Myc suppresses pdgf-betar mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-betar mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-betar mRNA levels plays an important role in the regulation of basal pdgf-betar expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-betaR.

Radiolabel tracer techniques were used to follow the distribution of nerve growth factor (NGF) and other neuromodulatory factors after intraventricular injection. Autoradiography showed that shortly after intraventricular injection of radio-iodinated NGF (125I-NGF), substantial amounts of radioactivity had penetrated the ventricular wall surfaces; this binding was transient and nonspecific. The 125I-NGF was progressively cleared from the central nervous system (CNS), presumably via the flow of cerebrospinal fluid (CSF) into the blood. A relatively small proportion of the injected 125I-NGF was taken up by NGF receptor-positive neurons in the CNS. Retrograde accumulation of radiolabel was observed within the basal forebrain cholinergic neurons at 5 hours after intraventricular injection. Labeling intensity was maximal at 18 hours and much reduced by 30 hours. This labeling was blocked by co-injection of an excess of unlabeled NGF. Specific and saturable retrograde labeling was also observed within other NGF receptor-bearing neurons, including the prepositus hypoglossal nucleus and the raphe obscurus nucleus. When epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-I), or IGF-II was radiolabeled and injected intraventricularly, specific labeling of neurons was observed for 125I-IGF-II and 125I-LIF within separate subpopulations of the dorsal and medial raphe. No retrograde accumulation within neurons was observed for EGF, TGF-beta 1, PDGF-AA, PDGF-BB, or IGF-I. This study describes an in vivo method for identifying putative neuromodulatory factors and their responsive neurons.

Insulin-like growth factor-I (IGF-I), a prevalent growth factor secreted by bone cells, has important effects on bone remodeling. Hormones are known to regulate the synthesis of skeletal IGF-I, but there is limited information about the actions of growth factors on IGF-I synthesis. We tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF beta 1), and platelet-derived growth factors (PDGF) AA and BB on IGF-I mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (Ob) cells from 22-day-old fetal rat calvariae. Steady state IGF-I mRNA levels were determined by Northern blot analysis, and IGF-I concentrations were determined in acidified and fractionated culture medium by a specific RIA. Treatment of Ob cells with bFGF at 0.06-6 nM, TGF beta 1 at 0.04-4 nM, and PDGF BB at 0.3-3.3 nM caused a dose-dependent decrease in steady state IGF-I mRNA. A smaller effect was observed with PDGF AA. The effect was initially observed after 6-8 h of treatment and was maximal after 16 h. Treatment with bFGF at 0.6-6 nM, TGF beta 1 at 0.4-4 nM, and PDGF BB at 0.3-3.3 nM for 24 h decreased IGF-I polypeptide concentrations by 40-80%. The effects of bFGF, TGF beta 1, and PDGF BB and AA on IGF-I mRNA were independent of protein synthesis and cell division, as they were observed in the presence and absence of cycloheximide at 3.6 microM or hydroxyurea at 1 mM. Similarly, their inhibitory actions on immunoreactive IGF-I were not prevented by hydroxyurea. In conclusion, bFGF, TGF beta 1, PDGF BB, and, to a lesser extent, PDGF AA decrease skeletal IGF-I synthesis by reducing IGF-I transcript levels, and this effect may contribute to their actions on selected aspects of Ob cell function.

Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca(2+)](i)were observed in single human VSMCs migrating on type I collagen. Such [Ca(2+)](i)transients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca(2+)](i) with BAPTA led to accumulation of (beta)1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca(2+)](i) transients in the rapid, serum-dependent migration of VSMCs. These [Ca(2+)](i)transients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.

Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by arterial smooth muscle cells (ASMCs). This proteoglycan accumulates in topographically defined regions of atherosclerotic lesions and may play a role in the development of this disease. However, little is known about whether decorin has specific effects on the cellular events that contribute to atherosclerotic lesion formation. In the present study, rat ASMCs were transduced with a retroviral vector (LDSN) that carries the bovine decorin gene. Compared with vector control cells (LXSN), these cells constitutively overexpress decorin, as verified by Northern and Western analysis and by metabolic labeling. Experiments were performed to examine the responsiveness of decorin-overexpressing rat ASMCs to platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1), 2 growth factors that affect cell proliferation and extracellular matrix production in atherosclerosis. Decorin-overexpressing cells had decreased [(3)H]thymidine incorporation into DNA and increased the levels of the cyclin-dependent kinase inhibitors p21 and p27 in the first 24 hours of response to serum and PDGF-BB. However, these effects of decorin were not apparent at 48 or 72 hours after plating and did not result in reduced growth of decorin-overexpressing cells in response to serum and PDGF-BB. In contrast, the growth response of decorin-overexpressing ASMCs to TGF-beta1, as well as the expression of TGF-beta1-responsive genes, such as plasminogen activator inhibitor-1 and versican (an extracellular matrix proteoglycan), was diminished. These results indicate that decorin selectively inhibits the responsiveness of rat ASMCs to TGF-beta1 and suggests that the induction of constitutive decorin overexpression by ASMCs in vivo may have therapeutic value in the inhibition of TGF-beta1-mediated effects on the development of atherosclerotic lesions.

The aim of this study was to investigate whether growth factors essential for fracture healing are substantially increased in the immediate aftermath following reaming of the intramedullary cavity for stabilisation of femoral shaft fractures. Consecutive adult patients whose femoral shaft fractures stabilised with either reamed (10 patients) or unreamed (10 patients) intramedullary nailing were studied. Peripheral blood samples and samples from the femoral canal before and after reaming and nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor-BetaBeta (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-I (IGF-I), Transforming Growth Factor beta 1 (TGF-beta1) and Bone Morphogenetic Protein-2 (BMP-2) was measured. The mean age of the twenty patients who participated in the study was 38 years (range 20-63). Reaming substantially increased all studied growth factors (p<0.05) locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-I was increased by 31.5% and TGF-beta1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF, TGF-beta1 remained unchanged while the levels of IGF-I decreased by 10%. The levels of these mediators in the peripheral circulation were not altered irrespectively of the nail insertion technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. This study indicates that reaming of the intramedullary cavity is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules.

In the present study we measured the level of PDGF receptor expression by chicken myoblasts and the effect of the three different PDGF isoforms (AA, AB, BB) on DNA synthesis by myoblasts. We examined PDGF receptor expression and function on clonally derived myoblasts in order to eliminate contaminating fibroblasts which are present in myogenic cultures and which bind PDGF. Furthermore, since we have previously shown that fetal myoblasts are replaced with adult myoblasts during late chicken embryogenesis, we compared PDGF receptor expression and function on myoblasts from Embryonic Day 10 (E10, mid development) and from Embryonic Day 19 (E19, late development). We found that all myogenic clones from late embryos (E19) express many receptors for PDGF-BB, far fewer receptors for PDGF-AB, and even fewer, if any, receptors for PDGF-AA. Myoblast clones derived from E10 were more heterogeneous in their PDGF binding pattern ranging from clones similar to E19 clones to clones having very few PDGF binding sites. We also found that both PDGF-AB and PDGF-BB can promote DNA synthesis by clonally derived chicken myoblasts maintained in 2.5% fetal bovine serum whereas PDGF-AA has no detectable effect. Finally, we observed that primary myogenic cultures from E10 and E19 differ strikingly in levels of PDGF binding; E19 cultures bind much more PDGF than do E10 cultures. We conclude that PDGF can enhance the proliferation of chicken myoblasts and that myoblasts responsive to PDGF are more frequent in late than in mid stages of development. We propose that PDGF may be a modulator of myogenesis of adult but not fetal myoblasts.

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-micro m perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGF(BB), PDGF(AA), and PDGF(AB) were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE(2), formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

Morphine stimulates tumor angiogenesis and cancer progression in mice. We examined if morphine influences endothelial-pericyte interaction via platelet-derived growth factor-BB (PDGF-BB) and PDGF receptor-β (PDGFR-β). Clinically relevant doses of morphine stimulated PDGF-BB secretion from human umbilical vein endothelial cells and activated PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in human pericytes. These in vitro effects of morphine were translated into promotion of tumor angiogenesis in a transgenic mice model of breast cancer when treated with clinically used dose of morphine. Increased vessel-associated immunoreactivity of desmin and PDGFR-β was observed on pericytes in tumors of morphine-treated mice. These data suggest that morphine potentiates endothelial-pericyte interaction via PDGF-BB/PDGFR-β signaling and promotes tumor angiogenesis, pericyte recruitment, and coverage of tumor vessels. We speculate that morphine may impair the effectiveness of antiangiogenic therapy by influencing vascular pericyte coverage.

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

This study examined the role of mitogen-activated protein (MAP) kinase in PDGF-BB-induced proliferation and gene expression of human mesangial cells (MC). PDGF-BB stimulation of MC increased mRNA for transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and increased the cell numbers. To inhibit activation of extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, MC were infected with recombinant adenovirus containing dominant-negative mutants of ERK, JNK, and p38 (Ad-DN-ERK, Ad-DN-JNK, Ad-DN-p38, respectively), respectively. Infection of MC with Ad-DN-ERK or Ad-DN-JNK inhibited PDGF-BB-induced increase in [(3)H]thymidine incorporation and cell numbers, whereas Ad-DN-p38 did not. Ad-DN-ERK inhibited MCP-1 and PAI-1 mRNA expression in MC, but not TGF-beta1. Ad-DN-JNK and Ad-DN-p38 inhibited TGF-beta1 and MCP-1 mRNA expression, but not PAI-1. The inhibition of activator protein-1 (AP-1) in MC, by adenovirus containing dominant-negative mutant of c-Jun (Ad-DN-c-Jun), inhibited PDGF-BB-induced cell proliferation and TGF-beta1, MCP-1, and PAI-1 expressions. Furthermore, Ad-DN-JNK or Ad-DN-p38, but not Ad-DN-ERK, attenuated PDGF-BB-induced AP-1 activation in MC, indicating the involvement of JNK and p38 in AP-1 activation. Our results indicated that ERK and JNK, but not p38, participated in PDGF-BB-induced MC proliferation. PDGF-BB-induced expression of TGF-beta1 was mediated by JNK and p38, MCP-1 expression was through ERK, JNK, and p38, whereas PAI-1 expression was due to only ERK. AP-1 activation, which was partially due to JNK and p38 activations, was involved in MC proliferation and these three gene expressions. Thus, three MAP kinases seem to contribute to progression of glomerular disease via different molecular mechanisms.

Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.

Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF beta-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4, 5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.