Involvement of the immunological mechanisms in atherogenesis has recently been suggested by immunohistological detection of macrophages and T lymphocytes in atherosclerotic lesions. In the present study, we have investigated the regulatory effect of interferon-gamma (IFN-gamma), a cytokine secreted by activated T cells, on the production and secretion of platelet-derived growth factor (PDGF) from macrophages in culture. The human monocytic leukemia cell line, THP-1, was treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce macrophage differentiation and PDGF production, and then various doses of recombinant human IFN-gamma (0-1000 I.U./ml) were added to the culture. After 48 h, the conditioned medium and the cells were harvested and analyzed for PDGF production. PDGF-dependent mitogenic activity in the conditioned medium, estimated by neutralization of mitogenic activity with anti-PDGF antibody, was suppressed by IFN-gamma treatment. Radioimmunoassays for PDGF also revealed a decrease in both PDGF-AA and -BB in the conditioned medium with IFN-gamma treatment, whereas neither total cell DNA as an indication of cell number nor overall protein synthesis based on [3H]leucine incorporation were decreased. Northern analysis of total RNA extracted from the cells demonstrated that IFN-gamma suppressed the level of PDGF mRNA. Analysis of mRNA degradation in the presence of actinomycin D demonstrated that the decrease in PDGF mRNA was not due to enhanced degradation of mRNA. A similar inhibitory effect of IFN-gamma on PDGF mRNA levels was also found in monocyte-derived macrophages cultured in the presence of granulocyte-macrophage colony stimulating factor. These results suggest that IFN-gamma modulates production and secretion of PDGF from macrophages and that the functions of macrophages in atherogenesis may be regulated by the cellular interactions between T cells and macrophages through the action of cytokines such as IFN-gamma.

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF β and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct "proximal" signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.

Angiotensin II (Ang II) may induce arterial hypertrophy either directly or through an increase in arterial pressure. To separate these 2 mechanisms, rats were implanted with osmopumps delivering either Ang II (100 ng x kg-1 x min-1) or saline. 5-Bromo-2'-deoxyuridine (BrdU) was delivered to both groups by osmopump (2.5 microg x kg-1 x min-1). Half of the rats in each group were given minoxidil (9 mg x kg-1 . d-1) in their drinking water. After 14 days, systolic blood pressure was 117+/-2, 124+/-3, and 115+/-2 mm Hg in the control, Ang II-minoxidil, and minoxidil groups, respectively, and 181+/-6 mm Hg in the Ang II group (P<0.05). After perfusion-fixation, the thoracic aorta, carotid artery, small mesenteric artery, external spermatic artery, and kidneys were harvested, paraffin-embedded, and used for morphological measurements, immunohistochemistry for BrdU, and in situ hybridization with a 35S-labeled riboprobe for platelet-derived growth factor-A chain (PDGF-A) mRNA. The walls of the aorta and carotid arteries hypertrophied in the Ang II group only. There were no significant morphological differences in the small arteries. BrdU was negative in all arteries but positive in the renal tubules. Expression of PDGF-A was elevated 8-fold in the thoracic aorta of the Ang II group (P<0.05). These results show that (1) arterial hypertrophy from Ang II infusion occurs in response to elevated arterial pressure, (2) hypertrophy was not associated with hyperplasia or polyploidy of vascular smooth muscle cells, and (3) PDGF-A expression correlated with elevated pressure and arterial wall hypertrophy.

BACKGROUND

Venous and combined malformations are slow-flow haemodynamically inactive lesions that are present at birth and worsen slowly with advancing age, showing no tendency towards involution. The pathogenesis of vascular anomalies has not been fully elucidated, but their formation and progression are closely related to angiogenesis. Localized intravascular coagulation associated with venous or combined malformations is characterized by low fibrinogen, high D-dimers, and normal platelet count.

OBJECTIVE

To assess the relationship of angiogenic factors with prothrombotic and endothelial damage/dysfunction markers in patients with extensive slow-flow vascular malformations.

METHODS

A 2-year study (2005-2007) included 31 consecutive patients with extensive slow-flow vascular malformations from one centre.

RESULTS

Serum levels of the endothelial receptor tyrosine kinase TIE-2, matrix metalloproteinase (MMP)-9 and angiopoietin (Ang)-2 and plasma levels of D-dimer, plasminogen activator inhibitor type 1 (PAI-1), tissue-type plasminogen activator and von Willebrand factor (vWf) were significantly increased in patients compared with healthy controls, whereas serum levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, MMP-2, Ang-1, platelet-derived growth factor (PDGF)-AB and PDGF-BB were significantly decreased in patients compared with controls. A strong positive correlation was present between Ang-1 and PDGF-AB levels (r = 0.63, P < 0.001), between PDGF-AB and PDGF-BB levels (r = 0.67, P < 0.001), and between fibrinogen and PAI-1 levels (r = 0.41, P = 0.031). A strong negative correlation was present between Ang-1 and vWf levels (r = -0.48, P = 0.006), between D-dimer and fibrinogen levels (r = -0.71, P < 0.001), and between PDGF-AB and vWf levels (r = -0.42, P = 0.017).

CONCLUSIONS

These findings suggest that angiogenic, coagulation and endothelial damage/dysfunction markers are possibly linked in pathogenesis of extensive slow-flow vascular malformations, and might have therapeutic implications.

Neuronal and vascular cells share common chemical signals. Vascular endothelial growth factor (VEGF)-C and -D and their receptor VEGFR-3/Flt-4 mediate lymphangiogenesis, but they occur also in the brain. Quantitative RT-PCR of mouse brain tissues and cultivated cells showed that the VEGFR-3 gene is highest transcribed in postnatal brain and in glial precursor cells whereas VEGF-C and -D are variably produced by different neuronal and glial cells. In neurospheres (neural stem cells) VEGFR-3 was induced by differentiation with platelet-derived growth factor (PDGF). In functional studies with an A2B5- and nestin-positive, O4-negative murine glial precursor cell line, VEGF-C and -D stimulated phosphorylation of the kinases Erk1/2; this signal transduction was inhibited by UO126. Both peptides induced the proliferation of glial precursor cells which could be inhibited by UO126. Furthermore, VEGF-D considerably enhanced their migration into an open space in a wound-healing assay. These results show that VEGF-C/-D together with its receptor VEGFR-3 provides an auto-/paracrine growth and chemotactic system for glial precursors in the developing brain.

Neural stem cells (NSC) are capable of differentiating toward neuronal, astrocytic, oligodendrocytic and glial lineages, depending on their spatial location within the central nervous system (CNS). Although, a lot of knowledge has been gained in the understanding of differentiation-specific signaling in hematopoietic (HSC) and mesenchymal (MSC) counterparts, the molecular mechanisms underlying lineage commitment in NSCs are just beginning to be understood. Furthermore, it is not well comprehended as to how the specification of one cell lineage can result in the suppression of parallel pathways in the NSCs. Thus, a thorough understanding of various signal transduction cascades activated via cytokines and growth factors, and the confounding effects of different CNS microenvironments are critically required to determine the full potential of NSCs. Our knowledge on the clonogenic ability, differentiation potential, and the inherent plasticity in both HSCs and MSCs may facilitate the understanding of lineage commitment in the NSCs as well. The information available from the marrow-derived stem cells may be extrapolated toward the similar signaling pathways in the neural precursors. From a number of previous studies, it is apparent that four distinctly different subsets of ligand-receptor superfamilies are involved in determining the fate of NSCs. These include 1) the transforming growth factor type-beta-1 (TGF-beta1) and bone morphogenetic protein (BMP) superfamily; 2) the platelet-derived and epidermal (PDGF/EGF) growth factors; 3) the interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor (IL-6/LIF/CNTF) superfamily; and 4) the EGF-like Notch/Delta group of extracellular ligands. Ligand binding to the cell surface receptor activates the receptor's cytosolic catalytic domain and/or the receptor-associated protein-kinases, which in turn activate intracellular second messengers and different sets of transcription factors. Transcription factor oligomerization, nuclear localization, followed by their recognition of DNA elements, leads to the expression of lineage-specific genes. Association between different groups of transcription factors can also regulate their ability to transcriptionally activate different genes. The limited availability of coactivators and cosuppressors, which can sequester the transcription factor complexes toward or away from a specific gene locus, further adds to the complexity in the cross talk between different signaling cascades. Both concerted actions of temporally regulated signals and convergent effects of different signaling cascades can thus ultimately precipitate the phenotypic changes. It is beginning to be realized that in addition to the cytokines and growth factors, cell-to-cell and cell-to-extracellular matrix (ECM) interactions, are also important within the molecular scenario linked to both proliferation and differentiation of the stem cells. The cell surface molecules, which include cell adhesion molecules (CAMs), integrins, selectins, and the immunoglobulins, are well known to regulate HSC and MSC commitment within different tissue microenvironments and may have direct implications in understanding the NSC cell fate determination within different regions of the brain.

Glucocorticoids are known to decrease wound healing by inhibiting the inflammatory response and collagen synthesis. In patients on steroids requiring emergency surgery, a safe agent that can be administered systemically is needed to reverse the deleterious effects of corticosteroids. TGF-beta and PDGF work topically but are not candidates for systemic administration. IGF-I and IGF-I:BP-3 are logical choices for systemic administration to improve wound healing. Both have been found in our laboratory to repair the corticosteroid-induced defect in wound healing when applied topically; the IGF-I:BP-3 complex gave significantly better results than IGF-I alone. Therefore, we asked whether these agents administered systemically could reverse the impaired wound healing caused by corticosteroids and whether the IGF-I:BP-3 complex was superior. Sprague-Dawley rats 350 g had 4 Hunt-Schilling wire mesh wound cylinders implanted s.c. on the back. Depo-Medrol (8 mg) was given s.c. at the time of surgery. Experimental rats were given daily s.c. injections of IGF-I or IGF-I:BP-3 (supplied by Celtrix Pharm, Santa Clara, CA) in PBS and 0.1% rat serum albumin, pH 6.0. The groups were: vehicle; IGF-I 125 micrograms/d; IGF-I:BP-3 complex containing 125 micrograms IGF-I/d. On post-op. day 17, the tissue in the wound cylinders was harvested and dried at 37 degrees C. Dry weight, DNA, total protein, and hydroxyproline (collagen) contents were obtained by our published procedures. Wound cylinder dry weight, DNA, total protein and hydroxyproline were increased by IGF-I 250%, 340%, 200% and 205%, respectively, over controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Epothilone D (Epo-D) is a paclitaxel-like microtubule-stabilizing agent that was isolated from the myxobacterium Sorangium cellulosum. Although Epo-D can inhibit proliferation in multiple tumor cell lines, the effect of Epo-D on neointimal hyperplasia after angioplasty has not been reported. The aim of the present study was to investigate the effects of Epo-D on neointimal hyperplasia using an in vivo rat carotid artery injury model. We demonstrated that local Epo-D treatment significantly reduced neointimal hyperplasia after in vivo rat carotid artery injury, and Epo-D potently inhibited DNA synthesis, cell cycle progression and cell proliferation after FBS- and PDGF-BB-stimulation; PDGF-BB has been identified as the most potent growth factor for stimulating the proliferation of activated rat aortic smooth muscle cells (RASMCs). To clarify the specific effects of Epo-D on cell cycle machinery, we examined its effects on cyclin-dependent kinase (CDK)2, CDK4, cyclin E, p27, and retinoblastoma (Rb) proteins as cell cycle-related proteins in cellular lysates from PDGF-BB-stimulated RASMCs. Epo-D treatment significantly decreased the level of CDK2 protein, but did not change the levels of CDK4 and cyclin E proteins. Furthermore, Epo-D inhibited the phosphorylation of Rb, a key regulator of the G1 to S phase transition in the cell cycle. These findings suggest that Epo-D may regulate the cell cycle G1-checkpoint proteins as its major molecular mechanism for inhibiting neointimal hyperplasia after in vivo rat carotid artery injury.

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.

Previous studies have demonstrated that interleukin-1 (IL-1) stimulates fibroblast growth (Schmidt, J. A., S. B. Mizel, D. Cohn, and I. Green. 1982. J. Immunol. 128:2177-2182) and binds to specific, high affinity receptors of BALB/c3T3 cells (Bird, T. A., and J. Saklatval. 1986. Nature (Lond.). 324:263-265, 266-268). We have investigated the mechanism of fibroblast growth stimulation by IL-1. Addition of fibroblast growth factor derived from platelets (PDGF) to a quiescent culture of BALB/c3T3 cells produced 8-10-fold increase in DNA synthesis during 24-h incubation. The cellular action of PDGF was mediated through competence induction and required synergistic action of plasma-derived factors for full mitogenic activity. When tested at a wide range of concentrations (0.1-100 pM), natural IL-1 or recombinant IL-1 produced only a maximum of 5-10% of DNA synthesis elicited in response to PDGF or serum. Induction of DNA synthesis required continuous presence of IL-1 and did not exhibit synergism with plasma. Competence induction and mitogenic stimulation by PDGF was associated with early induction of proteins P32, P38, P46-48, P75, and changes in cytoskeletal organization. Examination of these early cellular changes showed that IL-1 did not produce similar induction of cellular proteins and the morphological changes associated with growth stimulation. These results suggest that the mode of IL-1 action on BALB/c3T3 was not through competence induction. When IL-1 was added to cells rendered competent by brief exposure to PDGF, 10-15% additional DNA synthesis occurred during the first 24 h. Extended incubation of PDGF-treated cells in the presence of IL-1 revealed that the stimulation by IL-1 occurred predominantly during the subsequent cycle of DNA replication, wherein DNA synthesis reached three- to fivefold higher than the untreated cultures. We conclude (a) IL-1 alone is not a potent mitogen for BALB/c3T3 cells, and does not bring cells out of the growth arrest Go phase, (b) treatment with PDGF renders the cells more responsive to IL-1, (c) part of the IL-1 action on competent cells may be characterized as progression inducing activity, further, (d) our results indicate that action of IL-1 on PDGF-treated cells produces sustained DNA synthesis for an extended period, perhaps by preventing the entry of cells into growth arrest Go phase.

The transcription and expression of platelet-derived growth factor (PDGF) receptors (PDGFRs) is down-regulated as a consequence of entry into the replicative cell cycle (Vaziri, C., and Faller, D. V. (1995) Mol. Cell. Biol. 15, 1244-1253). In this study, we have investigated the expression of PDGFRs during terminal differentiation, a process in which cells exit from the cell cycle. When treated with appropriate hormonal stimuli, 3T3-L1 fibroblasts initiate a differentiation program resulting in conversion to lipid-accumulating, adipocyte-like cells. Pre-adipocytes express amounts of PDGFalphaR and PDGFbetaR mRNA and protein that are similar to levels expressed in other murine 3T3 fibroblasts. In contrast, the expression of both alpha and beta receptor transcripts is greatly reduced in differentiated 3T3-L1 cells. The loss of PDGFR mRNA following induction of differentiation precedes morphological conversion as well as the induction of many adipocyte-specific genes. The amounts of cell surface PDGFR protein diminish in parallel with the mRNA levels during differentiation, as shown by Western blotting and PDGF-binding assays. The reduced expression of PDGFRs does not reflect a general down-regulation of growth factor receptors, as expression of the type 1 FGFR is unaffected by terminal differentiation. The PDGFbetaR promoter drives strong expression of a luciferase reporter gene in pre-adipocytes, but not in differentiated cells, indicating that the decrease in PDGFR expression following induction of differentiation is a transcriptionally regulated event. Decreased PDGFR expression in differentiated cells is associated with impaired biological responsiveness to PDGF, as shown by reduced activation of mitogen-activated protein-kinase following PDGF stimulation, and decreased chemotactic responsiveness to PDGF. Our data suggest that PDGFR down-regulation is an important mechanism for reducing PDGF-responsiveness in terminally differentiated 3T3-L1 cells.

BACKGROUND

Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet.

METHODS

Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines.

RESULTS

The GF preparation contained a mean of 16.66, 2.04, 1.53, 72.19, 0.33, 48.59 and 0.44 ng/ml of PDGF-AB, -BB, -AA, TGF-beta1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0.1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates.

CONCLUSIONS

STANDARDIZED and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.

BACKGROUND

Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors alphavbeta3 and alphavbeta5, was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins alphavbeta3 and alphavbeta5 on RPE cells was examined.

METHODS

The effect of a cyclic integrin antagonist and a control peptide (0.01 microg/ml to 300 microg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H3-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors alphavbeta3 and alphavbeta5 was evaluated by flow cytometry.

RESULTS

The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1-10 microg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3-10 microg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1-10 microg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3 microg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors alphavbeta3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and alphavbeta5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold).

CONCLUSIONS

A selective inhibition of the integrin receptors alphavbeta3 and alphavbeta5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

BACKGROUND

Oxalate interaction with renal epithelial cells results in a program of events that include alterations in gene expression, re-initiation of DNA synthesis, cell growth and apoptosis. Our studies focused on understanding the mechanisms involved in the oxalate-induced re-initiation of the DNA synthesis. The effects of oxalate alone or in combination with epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were investigated to determine whether oxalate utilized the p42/44 mitogen activated protein (MAP) kinase pathway, which is a common pathway used by a majority of the mitogens.

METHODS

LLC-PK1 cells (a renal epithelial cell line of porcine origin) were exposed to oxalate in the presence or absence of three established growth factors, EGF, insulin and PDGF, and of the transcription/translation inhibitors, actinomycin-D and cycloheximide. DNA synthesis was assessed by [3H]-thymidine incorporation. p42/44 MAP kinase activity was assessed by super-shift analysis as well as by immunocomplex kinase assay.

RESULTS

Exposure of growth-arrested LLC-PK1 cells to oxalate resulted in the re-initiation of the DNA synthesis was abolished by [corrected] pretreatment with transcription/translation inhibitors. Oxalate (1 mmol/L), EGF (50 ng/mL) and insulin (100 ng/mL) stimulated DNA synthesis in growth-arrested LLC-PK1 cells, while PDGF (50 ng/mL) had no effect. Effects of EGF and oxalate on DNA synthesis were additive. In contrast, oxalate and insulin had antagonistic effects on DNA synthesis. Additionally, oxalate did not activate the p42/44 MAP kinase pathway while EGF stimulated this pathway.

CONCLUSIONS

These findings demonstrate that oxalate does not activate the p42/44 MAP kinase pathway, and the effects of oxalate are mediated by pathways that are distinct from those of EGF, PDGF and insulin.

Recent studies have suggested the importance of phosphatidylcholine (PC) metabolism in growth factor-stimulated cells. In these cells, PC is hydrolyzed not only by PC-specific phospholipase C but also by phospholipase D (PLD). In the present investigation, we show that the simple addition of PC-hydrolyzing PLD from Streptomyces chromofuscus to the culture medium of vascular smooth muscle cells elicits choline release into the medium accompanied by the formation of phosphatidic acid. In the presence of ethanol, this treatment elicits a formation of phosphatidylethanol (PEt) at the expense of phosphatidic acid. Furthermore, we show here that exogenous addition of S. chromofuscus PLD induces a marked DNA synthesis in quiescent vascular smooth muscle cells. This DNA synthesis induced by S. chromofuscus PLD is, like platelet-derived growth factor (PDGF)-elicited DNA synthesis, largely dependent on the presence of insulin. In addition, S. chromofuscus PLD-induced PEt formation and DNA synthesis were not affected by protein kinase C down-regulation, whereas PDGF-induced PEt formation and DNA synthesis were significantly inhibited. These observations strongly suggest that protein kinase-dependent activation of PLD is involved in mitogenic signal in PDGF-stimulated cells and that exogenously added PLD acts as a competence factor in the same way as PDGF.

Endothelin-1 (ET-1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) stimulated thymidine incorporation with different efficiency (PDGF>>) EGF = ET-1) in rat myometrial cells. They also stimulated ERK activation, which culminated at 5 min and then declined to reach a plateau (at 45 min: EGF>> 90%, PDGF = 50%, and ET-1 < 10% of maximum). Inhibition and downregulation of PKC demonstrated that ERK activation at 5 min involved PKC delta and -zeta for ET-1 and PKC alpha plus another PKC isoform for PDGF. By contrast, the EGF response did not involve PKC. Stimulation of Ras was more important with EGF than with PDGF, with ET-1 being the weakest activator. The simultaneous incubation of the cells with EGF and ET-1 potentiated the ERK activation at 5 min and mimicked the plateau phase obtained with PDGF. Under these conditions thymidine incorporation was comparable to that induced by PDGF. Taken together, our results indicated that the kinetic profile of ERK activation and its impact on cell proliferation can be modulated by the differential involvement of PKC isoforms and the amplitude of Ras activation.

Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.

OBJECTIVE

The present study was designed to use an in vivo rabbit ear scar model to investigate the efficacy of systemic administration of endostatin in inhibiting scar formation.

METHODS

Eight male New Zealand white rabbits were randomly assigned to two groups. Scar model was established by making six full skin defect wounds in each ear. For the intervention group, intraperitoneal injection of endostatin was performed each day after the wound healed (about 15 d post wounding). For the control group, equal volume of saline was injected. Thickness of scars in each group was measured by sliding caliper and the scar microcirculatory perfusion was assessed by laser Doppler flowmetry on Days 15, 21, 28, and 35 post wounding. Rabbits were euthanatized and their scars were harvested for histological and proteomic analyses on Day 35 post wounding.

RESULTS

Macroscopically, scars of the control group were thicker than those of the intervention group. Significant differences between the two groups were observed on Days 21 and 35 (p<0.05). Scar thickness, measured by scar elevation index (SEI) at Day 35 post wounding, was significantly reduced in the intervention group (1.09±0.19) compared with the controls (1.36±0.28). Microvessel density (MVD) observed in the intervention group (1.73±0.94) was significantly lower than that of the control group (5.63±1.78) on Day 35. The distribution of collagen fibers in scars treated with endostatin was relatively regular, while collagen fibers in untreated controls were thicker and showed disordered alignment. Western blot analysis showed that the expressions of type I collagen and Bcl-2 were depressed by injection of endostatin.

CONCLUSIONS

Our results from the rabbit ear hypertrophic scar model indicate that systemic application of endostatin could inhibit local hypertrophic scar formation, possibly through reducing scar vascularization and angiogenesis. Our results indicated that endostatin may promote the apoptosis of endothelial cells and block their release of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), thereby controlling collagen production by fibroblasts. Blood vessel-targeted treatment may be a promising strategy for scar therapy.

The great majority of murine Balb/c-3T3 fibroblasts in density-inhibited, quiescent cultures disintegrate and die rapidly when cells are deprived of serum in the medium. Platelet-derived growth factor (PDGF, 5 ng/ml) used alone and insulin-like growth factor (IGF-1, 40 ng/ml) + epidermal growth factor (EGF, 10 ng/ml) prevent most of this cell death and all three factors used together protect close to all cells in the confluent monolayer as determined by counting trypsinized cell suspensions in a Coulter counter. IGF-1 used alone affords a high level of protection during the first 5 hours of incubation in serum-free medium but the protective effect declines subsequently unless EGF is also present. EGF alone has little protective activity. The survival-promoting activity of PDGF used alone or of PDGF + EGF + IGF-1 is not significantly decreased by selective inhibition of messenger precursor RNA transcription with 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB, 20 or 40 microM), which prevents G1 traverse of the cells mediated by the combination of the three growth factors. DRB also does not interfere with the early protective effect of IGF-1 + EGF, but decreases the late protective effect of this growth factor combination. DRB by itself decreases cell viability in the absence of growth factors or serum. In these experiments viability was assayed by neutral red uptake by using an automated microplate reader. Inhibition of protein synthesis with cycloheximide (CHX, 1 or 5 micrograms/ml) over a 20-hour period was associated with decreased survival of cells protected by IGF-1 + EGF or PDGF + EGF + IGF, but also with decreased survival of cells incubated in the absence of growth factors or serum. The decrease in survival was somewhat more marked when IGF + EGF was present than when PDGF + EGF + IGF-1 was present. Insulin (1,500 ng/ml) mimics the action of IGF-1 (40 ng/ml). The cell survival-enhancing activities of growth factors are concentration dependent. The evidence presented indicates that PDGF, EGF, and IGF-1 (or insulin) act through distinctive mechanisms in affording protection of cells against death. The short-term protective effects of the growth factors are independent of gene expression and may be mediated via metabolic events. Long-term protection may be dependent on gene expression, especially in the case of IGF-1 + EGF.

Chronic allograft nephropathy (CAN), a descriptive term denoting chronic scarring injury of the renal parenchyma and vasculature in allograft kidneys arising from various etiologies including chronic rejection, is the most common cause of late allograft failure, but mediators of this progressive injury largely remain unknown. We hypothesized that platelet-derived growth factor D (PDGF-D) and its specific receptor PDGF-Rbeta may be an important mediator in the pathogenesis of CAN and, hence, sought to identify its expression in this setting. Allograft nephrectomies demonstrating CAN, obtained from patients with irreversible transplant kidney failure (n = 15), were compared with renal tissues without prominent histopathological abnormalities (n = 18) and a series of renal allograft biopsies demonstrating acute vascular rejection (AVR) (n = 12). Antibodies to PDGF-D and PDGF-Rbeta were used for immunohistochemistry. Double and triple immunohistochemistry was used to identify cell types expressing PDGF-D. PDGF-D was widely expressed in most neointimas in arteries exhibiting the chronic arteriopathy of CAN and only weakly expressed in a small proportion of sclerotic arteries in the other 2 groups. Double and triple immunolabeling demonstrated that the neointimal cells expressing PDGF-D were alpha-smooth muscle actin-expressing cells, but not infiltrating macrophages or endothelial cells. PDGF-Rbeta expression evaluated in serial sections was localized to the same sites where neointimal PDGF-D was expressed. PDGF-Rbeta was expressed in interstitial cells more abundantly in the CAN group compared with the normal and AVR groups, without demonstrable colocalization of PDGF-D. PDGF-D is present in the neointima of the arteriopathy of CAN, where it can engage PDGF-Rbeta to promote mesenchymal cell migration, proliferation, and neointima formation. PDGF-D may engage the PDGF-Rbeta to promote interstitial injury in chronic allograft injury, but its sources within the interstitium were unidentified.

The relationships that exist between proliferative states and proteoglycan (PG) synthesis have been examined in monkey aortic smooth muscle cells in culture. These cells were made quiescent in medium containing low serum (0.1%) and stimulated to divide by addition of either nanogram quantities or platelet-derived growth factor (PDGF) or medium containing 5% serum. Incorporation of [35S]sulfate into PG was increased during the first 24 h of growth stimulation, and this increase appeared to be principally in the large chondroitin sulfate proteoglycan (CSPG). Furthermore, addition of p-nitrophenyl beta-D-xyloside, which perturbs PG metabolism, inhibits cells from proliferating, suggesting that PG may be involved in facilitating cell division. Inhibition of cell proliferation by heparin and/or TGF-beta also causes elevated levels of 35S-sulfate incorporation into PG by these cells. These studies indicate that proteoglycan metabolism is modulated as a function of the growth of arterial smooth muscle cells; however, it is still uncertain whether PG play a direct or indirect role in the control of cell growth.

Platelet-derived growth factor (PDGF) is a major polypeptide mitogen for cells of mesenchymal origin such as fibroblasts. Chondroitin sulfate chains (CS), which are abundant in the extracellular matrix have been shown to physically interact with PDGF-BB modulating its biological function. The aim of the present study was to examine the involvement of CS on PDGF-BB induced proliferative responses and receptor activation in human lung fibroblasts. The addition of exogenous free CS chains caused a significant downregulation of the PDGF-BB mediated mitogenic and chemotactic responses. Similar results were obtained by the increase of endogenous CS biosynthesis after beta-D-xyloside treatment. Furthermore, removal of the membrane-bound CS chains by selective enzymatic treatment significantly increased the proliferative capacity of human fibroblasts. Analysis of PDGF-R phosphorylation in the presence of CS or beta-D-xyloside, revealed a reduction of PDGF-Rbeta phosphorylation in the tyrosine residue 1021. These results demonstrate, for the first time, that CS either soluble or surface bound downregulates the mitogenic responses of PDGF-BB in normal human lung fibroblasts through the reduction of PDGF-Rbeta phosphorylation.

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-beta, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)-delta and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.

The purpose of this study is to investigate the early responses of human periodontal ligament cells attached to recombinant human platelet-derived growth factor-BB and bone morphogenetic protein-2 applied EDTA-demineralized dentin. One hundred and seventy-four root-planed flat dentin blocks were prepared from the mid-third of periodontally diseased human tooth roots. After demineralization with 24% EDTA (pH 7.02) 120 dentin blocks were treated with 0.5 and 1 microgram/ml rhPDGF-BB, 1 and 3 micrograms/ml rhBMP-2 and only MEM as control (24/group). Human periodontal ligament cells (HPLC) were seeded on these dentin surfaces and incubated. The alkaline phosphatase (ALP) activity and protein concentration of the attached cell were assessed at d 2, 4 and 7. Fifty-four dentin blocks were seeded with HPLC after application of 1 microgram/ml rhPDGF-BB, 3 micrograms/ml rhBMP-2 and MEM (18/group) and then incubated. At d 2, 4 and 7, the attached cells were stained and counted under light microscope. The results showed a significant increase of protein concentration and cell number in PDGF-BB treated groups than control (p < 0.05, p < 0.01) but not the ALP activity, and a significant increase of ALP activity was observed in BMP-2 treated groups than control (p < 0.05) but protein concentration and cell number remained almost the same over time. Thus, rhPDGF-BB and rhBMP-2 application to EDTA demineralized dentin surfaces promote the early human periodontal ligament cell responses by increasing cell proliferation and differentiation, respectively, which would ultimately enhance periodontal regeneration.