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PubMed1997

Platelet derived growth factor stimulates chondrocyte proliferation but prevents endochondral maturation.

Platelet-derived growth factor (PDGF) is a cytokine released by platelets at sites of injury to promote mesenchymal cell proliferation. Since many bone wounds heal by endochondral bone formation, we examined the response of chondrocytes in the endochondral lineage to PDGF. Confluent cultures of rat costochondral resting zone cartilage cells were incubated with 0-300 ng/mL PDGF-BB for 24 h to determine whether dose-dependent changes in cell proliferation (cell number and [3H]-thymidine incorporation), alkaline phosphatase specific activity, [35S]-sulfate incorporation, or [3H]-proline incorporation into collagenase-digestible protein (CDP) or noncollagenase-digestible protein (NCP), could be observed. Long-term effects of PDGF were assessed in confluent cultures treated for 1, 2, 4, 6, 8, or 10 d with 37.5 or 150 ng/mL PDGF-BB. To determine whether PDGF-BB could induce resting zone chondrocytes to change maturation state to a growth zone chondrocyte phenotype, confluent resting zone cell cultures were treated for 1, 2, 3, or 5 d with 37.5 or 150 ng/ml PDGF-BB and then challenged for an additional 24 h with 1,25-(OH)2D3. PDGF-BB caused a dose-dependent increase in cell number and [3H]-thymidine incorporation at 24 h. The proliferative effect of the cytokine decreased with time. PDGF-BB had no effect on alkaline phosphatase at 24 h, but at later times, the cytokine prevented the normal increase in enzyme activity seen in post-confluent cultures. This effect was primarily on the cells and not on the matrix. PDGF-BB stimulated [35S]-sulfate incorporation at all times examined, but had no effect on [3H]-proline incorporation into either the CDP or NCP pools. Thus, percent collagen production was not changed. Treatment of the cells for up to 5 d with PDGF-BB failed to elicit a 1,25-(OH)2D3 responsive phenotype typical of rat costochondral growth zone cartilage cells. These results show that committed chondrocytes can respond to PDGF-BB with increased proliferation. The effect of the cytokine is to enhance cartilage matrix production, but at the same time to prevent progression of the cells along the endochondral maturation pathway.

Alkaline Phosphatase↗
PubMed2005

PD98059 inhibited the activation of pancreatic stellate cells mediated by platelet-derived growth factor BB in rats.

To determine the biological effects of extracelluar signal regulated kinase (ERK) specific inhibitor PD98059 on pancreatic stellate cells (PSCs) activated by platelet-derived factor-BB (PDGF-BB), cultured rat PSCs were co-incubated at 37 DC for 24 h with 25 ng/ml PDGF-BB and different doses of PD98059 (ranging from 5 ng/ml to 40 ng/ml). Expression of pERK1 protein was detected by Western blot and collagen alpha1 ( I ) mRNA was tested by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that there were statistical differences in the expression of pERK1 protein in all groups. Expression of pERK1 protein was up-regulated in the group treated by PDGF-BB, and gradually down-regulated in the other groups treated by PD98059 of different doses. An excellent positive correlation was revealed between the inhibitory effect and PD98059 concentrations. It was also observed that the expression of collagen alpha1 ( I ) mRNA had similar response to pERK1. The level of collagen alpha1 ( I ) mRNA was the highest in the PDGF-BB group, and gradually reduced in the other groups treated by PD98059 of different doses. It is concluded that expression of pERK1 protein and collagen alpha1 ( I ) mRNA was up-regulated in rat PSCs activated by PDGF-BB. Meanwhile, PD98059 could inhibit PSCs activation mediated by PDGF. It is suggested that ERK1 protein plays an important role on PSCs activation mediated by PDGF signal pathway.

Animals↗
PubMed2001

Platelet-derived growth factor enhances granulopoiesis via bone marrow stromal cells.

Platelet-derived growth factor (PDGF), a growth factor for connective tissue cells, stimulates erythropoiesis and megakaryocytopoiesis in vitro but the effect of PDGF on granulocyte proliferation remains unknown. The effect of the recombinant human PDGF-BB isoform on granulopoiesis was investigated in this study. The results show that PDGF significantly stimulated murine colony-forming unit-granulocyte-monocyte (CFU-GM) proliferation in a dose-dependent manner (1 to 100 ng/mL) using murine bone marrow cells (n = 4). Maximum stimulation was obtained with 50 ng/mL of PDGF (P < .01). The effect of PDGF on murine CFU-GM proliferation was compared with that of interleukin (IL)-3, IL-6, granulocyte-monocyte colony-stimulating factor (GM-CSF), and acidic fibroblast growth factor (aFGF) at their optimal doses. The stimulating activity of PDGF was higher than that of aFGF but lower than that of IL-3, IL-6, or GM-CSF. There is no synergistic effect between PDGF and IL-3 or IL-6, but a significant enhancing effect was observed in IL-3 plus IL-6. PDGF also stimulated the growth of CFU-GM with CFU-megakaryocyte in the presence of bone marrow stromal cells. We also found that PDGF had similar a effect on human CFU-GM proliferation using bone marrow mononuclear cells (MNC). However, the increase in PDGF-stimulated CFU-GM proliferation was inhibited by anti-GM-CSF, anti-IL-3, and anti-IL-6 antibodies (n = 4), suggesting that endogenously produced GM-CSF, IL-3, and IL-6 may play a role in the PDGF-induced CFU-GM proliferation. Furthermore, PDGF (1 to 100 ng/mL) did not show any effect on CFU-GM proliferation when replacing bone marrow MNC with immunomagnetic selection-enriched CD34+ cells from human cord blood (n = 5; purity, 91% +/- 6.5%). This study indicates that PDGF may indirectly enhance CFU-GM proliferation by inducing the bone marrow stromal cells to produce GM-CSF, IL-3, or IL-6.

Adult↗
PubMed1999

Control of apoptosis of cardiovascular fibroblasts: a novel drug target.

Adequate control of survival or programmed cell death (apoptosis) of cardiovascular cells appears as an important drug target. While prevention of apoptotic death of cardiomyocytes has been assessed in detail, selective induction of apoptosis of vascular smooth muscle cells or fibroblasts could also be of relevance. Thus, induction of apoptosis of vascular smooth muscle cells by p65 NF-kappa B and Bcl-xL antisense oligonucleotides or p53 overexpression could be useful for limiting vascular lesions associated with restenosis. Although fibroblasts represent the majority of cardiac cells, few attempts were made to induce fibroblast apoptosis in disorders associated with excessive collagen deposition and fibrosis. It is hypothesized that early interference with fibroblast proliferation after myocardial infarction or inflammatory heart disease limits fibrosis which further impairs cardiac performance. A candidate approach could involve growth factor analogues which are known to induce fibroblast apoptosis when an incomplete growth stimulus persists.

Anticoagulants↗
PubMed2003

Growth factor releasing porous poly (epsilon-caprolactone)-chitosan matrices for enhanced bone regenerative therapy.

Drug releasing porous poly(epsilon-caprolactone) (PCL)-chitosan matrices were fabricated for bone regenerative therapy. Porous matrices made of biodegradable polymers have been playing a crucial role as bone substitutes and as tissue-engineered scaffolds in bone regenerative therapy. The matrices provided mechanical support for the developing tissue and enhanced tissue formation by releasing active agent in controlled manner. Chitosan was employed to enhance hydrophilicity and biocompatibility of the PCL matrices. PDGF-BB was incorporated into PCL-chitosan matrices to induce enhanced bone regeneration efficacy. PCL-chitosan matrices retained a porous structure with a 100-200 microm pore diameter that was suitable for cellular migration and osteoid ingrowth. NaHCO3 as a porogen was incorporated 5% ratio to polymer weight to form highly porous scaffolds. PDGF-BB was released from PCL-chitosan matrices maintaining therapeutic concentration for 4 week. High osteoblasts attachment level and proliferation was observed from PCL-chitosan matrices. Scanning electron microscopic examination indicated that cultured osteoblasts showed round form and spread pseudopods after 1 day and showed broad cytoplasmic extension after 14 days. PCL-chitosan matrices promoted bone regeneration and PDGF-BB loaded matrices obtained enhanced bone formation in rat calvarial defect. These results suggested that the PDGF-BB releasing PCL-chitosan porous matrices may be potentially used as tissue engineering scaffolds or bone substitutes with high bone regenerative efficacy.

Animals↗
PubMed2004

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor.

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05-0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05-0.001) and restored Akt phosphorylation in retinal pericytes. CONCLUSIONS/INTERPRETATION: Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.

Animals↗
PubMed2005

Health-related quality of life of diabetic foot ulcer patients and their caregivers.

AIMS/HYPOTHESIS: The effect of a foot ulcer on health-related quality of life (HRQoL) of patients with diabetes mellitus and their caregivers is unclear, and was therefore evaluated prospectively in this multicentre study. METHODS: HRQoL according to the 36-item health-related quality of life questionnaire (SF-36) of 294 patients (ulcer duration > or = 4 weeks) and 153 caregivers was analysed at baseline (time-point zero [T0]), once the ulcer was healed or after 20 weeks (time-point 1 [T1]), and 3 months later (time-point 2 [T2]). Patients with severe ischaemia were excluded. RESULTS: The mean age of the patients was 60 years, 72% were male, and time since diagnosis of diabetes was 17 years. Patients reported a low HRQoL on all SF-36 subscales. At T1, HRQoL scores in physical and social functioning were higher in patients with a healed vs a non-healed ulcer (p<0.05). At T2, these differences were larger, with higher scores for physical and social functioning, role physical and the physical summary score (all p<0.05). Within-group analysis revealed that HRQoL improved in different subscales in patients with a healed ulcer and worsened in patients with a persistent ulcer from T0 to T2 (all p<0.05). The caregivers of patients with a persisting ulcer had more emotional difficulties at T2. CONCLUSIONS/INTERPRETATION: Diabetic patients with a healed foot ulcer had a higher HRQoL than patients with a persisting ulcer. Healing of a foot ulcer resulted in a marked improvement of several SF-36 subscales 3 months after healing (from T0 to T2). HRQoL declined progressively when the ulcer did not heal. A diabetic foot ulcer appeared to be a large emotional burden on the patients' caregivers, as well.

Becaplermin↗
PubMed2000

[Degree of differentiation of chondrocytes and their pretreatment with platelet-derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo].

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.

Animals↗
PubMed2003

Cigarette smoke extract inhibits chemotaxis and collagen gel contraction mediated by human bone marrow osteoprogenitor cells and osteoblast-like cells.

Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-beta1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-beta1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis.

Becaplermin↗
PubMed2006

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO.

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)2(dppz)]2+. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)2(dppz)]2+ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer-TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L(-1), which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)2(dppz)]2+. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.

Aptamers, Nucleotide↗
PubMed1998

Mitogenic action of adenosine on osteoblast-like cells, MC3T3-E1.

The purpose of this study was to investigate the mechanisms by which adenosine stimulates proliferation of osteoblast-like cells, MC3T3-E1. Adenosine by itself induces the stimulation of cell proliferation and accentuates the mitogenecity of PDGFs (AA and BB homodimers) for the cells. 8-Cyclopentyl-1,3-dimethylxanthine (CPX), a nonselective adenosine receptor antagonist, partially inhibited adenosine-induced DNA synthesis in a competitive manner, suggesting that the mitogenic action of adenosine is, at least in part, mediated by xanthine-sensitive receptors. In pertussis-toxin (PTX)-pretreated cells, adenosine- but not PDGF-BB-stimulated DNA synthesis was partially inhibited, and CPX did not exert a further inhibitory effect, suggesting an involvement of PTX-sensitive G-protein downstream of CPX-sensitive receptor. When adenosine uptake was prevented with dipyridamole, the stimulation of proliferation by adenosine was not decreased at all, indicating that the CPX-insensitive part of adenosine action is not associated with the uptake of adenosine and subsequent incorporation into the nucleotide pool. Adenosine did not influence the basal level or the PDGF-BB-induced increase in [Ca2+]i. Since it is known that the cAMP pathway acts in inhibiting osteoblast proliferation, the mitogenic action of adenosine would be dependent on neither the cAMP pathway nor the phospholipase C/Ca2+ pathway. It has been concluded that adenosine exerts a mitogenic effect via two pathways at least, one mediated by xanthine-sensitive receptor and PTX-sensitive G-protein and the other through an unknown xanthine- and PTX-insensitive process.

Adenosine↗
PubMed2005

Wound healing enhancement: electroporation to address a classic problem of military medicine.

The major goal of wound healing biology is to determine how a wound can be induced to repair damaged tissue faster and more efficiently. Enhancement of dermal and epidermal regeneration is an extremely important goal for the treatment of many different types of wounds. Exogenous application of growth factors to the wound site has been shown to have potential to improve wound healing. Frequent applications of large amounts of growth factor have been required. This is because proteases in the wound quickly destroy peptide growth factor. Gene therapy has the potential to produce growth factors deep within the wound, where they can be effective as well as able to constantly replenish growth factor that is destroyed by peptidases. We have shown that application of plasmid DNA expression vectors directly into the wound is an inefficient modality. Electroporation, the application of an electrical field across cells to permeabilize the cell membrane has led us to explore the possibility of utilizing the technique to enhance transfection efficiency. We have identified electroporation parameters that improve the efficiency of DNA transfection in cutaneous wounds, and we have shown that electroporation itself does not impair wound healing. We are now on the threshold of exploring whether electroporation-assisted transfection with DNA plasmid expression vectors for growth factors will be an effective modality for enhancing cutaneous wound healing.

Animals↗
PubMed2002

Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation.

Activation of the local and systemic renin-angiotensin system is directly and indirectly involved in mechanisms of vascular remodeling during chronic hypertension. This study investigated the effect of angiotensin II (AII) on rat vascular smooth muscle cell (VSMC) migration towards platelet-derived growth factor-BB (PDGF-BB) in vitro. Pre-treatment with AII (1 microM) for 48 or 72 h induced a significant increase in PDGF-BB-directed migration by 77 +/- 21 % and 58 +/- 24 %, respectively (both p < 0.01). This effect was concentration dependent and inhibited by the selective angiotensin receptor type I (AT(1)) blocker DUP 753. PDGF-directed migration of VSMCs was significantly inhibited by antibodies against beta(3)-and beta(5)-integrins, indicating an important role of these integrins in VSMC migration. However, AII augmented migration was not accompanied by an increased expression of beta(3)- and beta(5)-integrin mRNA and protein levels in VSMCs. Inhibition of the mitogen-activated protein kinase ERK 1/2 with PD 98059 (30 microM) completely abolished the effect of AII on PDGF-BB-directed VSMC migration (p < 0.01). The proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are cytoskeleton-associated protein kinases participating in integrin-dependent signaling. Therefore, expression and phosphorylation of these kinases was determined 48 h after AII treatment, revealing a significant increase in Pyk2 and FAK protein levels (up to 2-fold, both p < 0.05) and increased phosphorylation of Pyk2 (2-fold, p < 0.05) and ERK 1/2 (4-fold, p < 0.05) as compared to controls. Furthermore, immunofluorescence and Western blot analysis demonstrated a translocation of Pyk2 from the plasma membrane to the cytosol, as well as a perinuclear enrichment of ERK 1/2 protein 48 h after AII treatment. In conclusion, our data suggest that changes in the levels of Pyk2 and ERK 1/2 phosphorylation, responsible for integrin-dependent signaling, as well as their subcellular translocation are important for the enhanced chemotactic response of VSMCs after AII pre-treatment.

Angiogenesis Inducing Agents↗
PubMed1998

Antimitogenic effects of vasodilatory prostaglandins in coronary artery smooth muscle cells.

Vasodilatory prostaglandins (PGI2, PGE2, PGE1) are known inhibitors of proliferation of vascular smooth muscle cells (SMC) after stimulation with mitogenic factors. However, endogenous prostaglandins do not prevent SMC proliferation subsequent to vessel injury in vivo. Since vascular cells produce large amounts of antiproliferative prostaglandins, especially subsequent to COX-2 expression, insufficient vascular PGI2 formation is not likely to explain the failure of endogenous prostaglandins to prevent excessive SMC growth. In this paper we demonstrate a rapid development of tolerance to PGI2 in SMC, resulting in diminished antiproliferative activity. These findings may not only be relevant for the control of SMC growth by endogenously synthesized prostaglandins but also for clinical use of PGI2 mimetics.

Animals↗
PubMed2001

Inhibition of rat cardiac fibroblast growth by cAMP--but not by cGMP-dependent protein kinase.

Cardiac fibroblasts play a critical role in the process of pathophysiological cardiac hypertrophy as the cell type responsible for fibrosis. Whereas many growth factors and hormones are thought to be involved, possible crosstalks between signal transduction pathways are not well defined. Therefore we investigated the influence of cAMP- and cGMP-dependent protein kinases (cAK, cGK) on platelet derived growth factor (PDGF) stimulated growth of primary cardiac fibroblasts from adult rats. We show here that PDGF-BB induced cell proliferation can be inhibited by activation of the endogenous cAK directly via the cAMP analog 5,6-DCL-cBIMPS as well as indirectly via the cAMP-elevating receptor agonist prostaglandin-E1 (PGE1). In contrast, activation of the endogenous cGK-I has no influence on cardiac fibroblast cell growth. The strength of the proliferation inhibition is dependent on the time course of cAK activation, i.e., longer activation with the cAMP analog results in stronger proliferation inhibition. No significant influence of cAK or cGK-I on Akt activation or on the short-term activation of the MAPK cascade was observed. In contrast, 5,6-DCI-cBIMPS treatment of cardiac fibroblasts causes an inhibition of long-term MAPK phosphorylation. A prolonged PGE1-dependent cAMP signal after addition of 3-isobutyl-1-methylxanthine (IBMX) blocks the second long-term MAPK phosphorylation as well. Therefore the suppression of MAPK long-term phosphorylation, four to eight hours after PDGF-BB stimulation, appears to play the major role in inhibition of cardiac fibroblast proliferation by cAK activators.

Animals↗
PubMed2003

Activation of p38 MAPK suppresses matrix metalloproteinase-1 gene expression induced by platelet-derived growth factor.

p38 mitogen-activated protein kinase (MAPK) regulates matrix metalloproteinase-1 (MMP-1) gene expression bidirectionally depending on the induction. We sought to determine whether cytokines related to the regulation of extracellular matrix could activate p38 MAPK in dermal fibroblasts. We determined p38 MAPK phosphorylation/activation in dermal fibroblasts stimulated with platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta or interleukin-4. Induction of MMP-1 mRNA by PDGF-BB was enhanced in the presence of a specific inhibitor of p38 MAPK, suggesting that p38 MAPK would function as a negative regulator of the MMP-1 mRNA level. We then determined which isoforms of p38 MAPK expressed in dermal fibroblasts were responsible for the downregulation of the MMP-1 mRNA level. Overexpression of p38beta2, but not of p38alpha, significantly decreased PDGF-BB-induced MMP-1 promoter activity, although PDGF-BB activated signaling pathways to both p38alpha and p38beta2. Taken together, the results of this study indicate that p38beta2 can function as a negative regulator of MMP-1 induced by PDGF-BB in vitro, suggesting that activation of p38beta2 might contribute to the pathogenesis of cutaneous fibrosis.

Animals↗
PubMed1999

Effect of heparin on human corneal fibroblast proliferation in vitro with and without growth factor stimulation.

BACKGROUND: The outcome of keratorefractive procedures such as PRK and LASIK is limited by the wound-healing process in the corneal stroma, which gives rise to complications such as haze formation and regression. The proliferation and matrix synthesis of corneal stromal fibroblasts is the central element of the wound-healing process. In order to develop new therapeutic strategies to reduce wound-healing intensity, we investigated the effect of heparin on the proliferation of cultured human corneal stromal fibroblasts (HCF) alone and in the presence of growth factors. METHODS: Primary cultures of HCF were established using epithelium and endothelium-free explants. Secondary cultures of HCF (first passage), cultured in WM/F12 supplemented with 10 microg/ml transferrin and 10 microg/ml thyroglobulin (LR-1 medium), 1% fetal calf serum (FCS) and 10% FCS were used to determine the effect of heparin on the proliferation of HCF in concentrations ranging from 12.5 microg/ml to 5000 microg/ml. Cell number was determined using the CASY 1 cell counter system. Modulation of HCF proliferation by heparin (50 microg/ml and 2000 microg/ml) was also investigated under serum-free conditions and in the presence of bFGF, EGF and PDGF-BB. RESULTS: Addition of heparin led to a dose-dependent inhibition of proliferation after 6 days of incubation, which was statistically significant for 500-5000 microg heparin/ ml (FCS 1%) and for 200-5000 microg heparin/ml (FCS 10%). IC50 values for this effect were determined to be approximately 700 microg heparin/ml. When cultured under serum-free conditions (LR-1), a significant reduction of cell number was only observed with 5000 microg heparin/ml. There was no significant modulation of PDGF-BB-, bFGF-, or EGF-stimulated cell proliferation by heparin at concentrations of 50 microg/ml and 2000 microg/ml after 6 days of incubation. CONCLUSION: Our observations indicate that heparin can inhibit proliferation of HCF effectively. The results of the present study could eventually pave the way to prevent anterior stromal haze formation and regression after keratorefractive surgery.

Anticoagulants↗
PubMed2003

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans.

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A(L) and B(L)) or short (A(S) and B(S)) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA(L) and PDGF-BB(S) isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.

Animals↗