Vascular proliferative disorders are characterized by migration and proliferation of vascular smooth muscle cells (SMCs), loss of expression of SMC phenotype, and enhanced extracellular matrix synthesis (e.g., type I collagen). We report here that bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor-beta (TGF-beta) superfamily, is capable of inhibiting both serum-stimulated and growth factor-induced (platelet-derived growth factor [PDGF-BB] and TGF-beta1) cell growth as measured by (3)H-thymidine uptake into DNA synthesis and cell number in primary human aortic smooth muscle (HASM) cell cultures. Concomitantly, addition of BMP-7 stimulates the expression of SMC-specific markers, namely alpha-actin and heavy chain myosin as examined by RT-PCR and Northern blot analyses. The collagen type III/I ratio that becomes lower with the transdifferentiation of SMCs into myofibroblasts is also maintained in BMP-7-treated cultures as compared to untreated controls. Studies on the mechanism of action indicate that BMP-7 treatment inhibits cyclin-dependent kinase 2 (cdk-2) that was stimulated during PDGF-BB-induced proliferation of SMCs and upregulates the expression of the inhibitory Smad, Smad6, which was shown to inhibit TGF-beta superfamily signaling. These results collectively suggest that BMP-7 maintains the expression of vascular SMC phenotype and may prevent vascular proliferative disorders, thus potentially acting as a palliative after damage to the vascular integrity.

Platelet-derived growth factor BB (PDGF) stimulates DNA synthesis through a mechanism that is at least partially dependent upon Src family tyrosine kinases, although the signal transduction pathway downstream of Src is poorly understood. We have studied the signaling between Src and different protein kinase C (PKC) isoforms and its possible role in the regulation of PDGF-stimulated DNA synthesis. We found that Src promoted the tyrosine phosphorylation of PKCdelta, and its subsequent degradation. Enforced expression of PKCdelta inhibited PDGF-stimulated DNA synthesis, whereas expression of PKCalpha and PKCepsilon did not, a finding consistent with a model in which PKCdelta negatively regulates G1-to-S-phase progression. We used mutagenesis to map a critical Src phosphorylation site on PKCdelta to tyrosine 311. A mutant form of PKCdelta in which tyrosine 311 was replaced with phenylalanine (Y311F) was more stable in the presence of Src, suggesting that Src-induced degradation was a direct result of PKCdelta tyrosine phosphorylation. We conclude that PKCdelta is downstream of Src but is unlikely to play a positive role in the signaling pathway by which Src promotes DNA synthesis.

Secreted modular calcium-binding protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we, found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed after mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of platelet-derived growth factor-beta receptor (PDGFbetaR), a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and basic fibroblast growth factor). Conversely, SMOC-2 ablation by using small interfering RNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells, and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis after SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of mitogen-activated protein kinase and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, integrin-linked kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control.

BACKGROUND

Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.

RESULTS

Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.

CONCLUSIONS

We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

BACKGROUND

The objective of this study was to explore whether individual variations in the concentration of growth factors (GFs) influence the biologic effects of platelet-rich plasma (PRP) on human mesenchymal stem cells (HMSCs).

METHODS

The concentrations of 7 representative GFs in activated PRP (aPRP) were measured using ELISA. The effects of PRP on the proliferation and alkaline phosphatase (ALP) activity of HMSCs were examined using several concentrations of aPRP from 3 donors; the relationships between the GF levels and these biologic effects were then evaluated using 10% aPRP from 5 subgroups derived from 39 total donors. HMSCs were cultured in DMEM with the addition of aPRP for 4 or 12 days; then, DNA content and ALP activity were measured.

RESULTS

The quantity of DNA increased significantly at a 10% concentration of aPRP, but the ALP activity was suppressed at this concentration of aPRP. The GF concentrations varied among donors, and 5 subgroups of characteristic GF release patterns were identified via cluster analysis. DNA levels differed significantly between groups and tended to be higher in groups with higher concentrations of transforming growth factor-beta1 (TGF-β1) and platelet-derived growth factors (PDGFs). DNA quantity was positively correlated with TGF-β1 concentration, and was negatively correlated with donor age. ALP activity was negatively correlated with PDGF-BB concentration.

CONCLUSIONS

The varying GF concentrations may result in different biologic effects; thus, individual differences in GF levels should be considered for reliable interpretation of the biologic functions and standardized application of PRP.

Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF alphaalpha-, alphabeta-, and betabeta-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM alpha-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM alpha-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF beta-receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE(-/-)) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow.

OBJECTIVE

The goals of the present study were to investigate the mechanism of hypoxia-mediated chemoresistance in liver cancer cells and tumorigenic hepatic progenitor (oval) cells and to determine whether disrupting an Akt/hypoxia-inducible factor-1alpha (HIF-1alpha)/platelet-derived growth factor (PDGF)-BB autocrine loop can enhance chemotherapeutic efficacy in hypoxia.

METHODS

Five hepatocellular carcinoma (HCC) cell lines and two hepatic progenitor cell lines were treated in vitro with cisplatin under both normoxic and hypoxic conditions. To generate ischemic hypoxia for tumor cells in vivo, hepatic artery ligation was applied to an orthotopic HCC model. Cisplatin and YC1, which is a HIF-1alpha inhibitor, were administered by portal vein and intratumoral injections, respectively.

RESULTS

Cell viability was higher under hypoxic than normoxic conditions. HIF-1alpha and Akt were up-regulated under hypoxic conditions, forming an autocrine signaling loop with PDGF-BB. Akt/HIF-1alpha/PDGF-BB signaling regulated Akt to confer cisplatin resistance to HCC cell lines in vitro. This autocrine signaling loop also contributed to chemoresistance in the tumorigenic hepatic progenitor cell line PIL2 under hypoxic conditions but not in the nontumorigenic cell line PIL4. In an orthotopic HCC model, combining blockade of HIF-1alpha activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival.

CONCLUSIONS

Blockade of Akt/HIF-1alpha/PDGF-BB autocrine signaling could enhance the chemosensitivity of liver cancer cells and tumorigenic hepatic progenitor cells under hypoxic conditions and thus provide an effective therapeutic strategy for HCC.

For the purpose of obtaining high bone forming efficacy, development of chitosan was attempted as a tool useful as a scaffolding device. Porous chitosan matrices, chitosan-poly(L-lactide) (PLLA) composite matrices and chitosan coated on PLLA matrices were dealt with in this research. Porous chitosan matrix was fabricated by freeze-drying and cross-linking aqueous chitosan solution. Porous chitosan matrix combined with ceramics and constituents of extracellular matrices were prepared and examined for their bone regenerative potential. Composite porous matrix of chitosan-PLLA was prepared by mixing polylactide with chitosan and freeze-drying. All chitosan based devices demonstrated improved bone forming capacity by increasing mechanical stability and biocompatibility. Release of platelet-derived growth factor-BB (PDGF-BB) from these matrices exerted significant osteoinductive effect in addition to the high osteoconducting capacity of the porous chitosan matrices. The hydrophobic surface of PLLA matrices was modified by chitosan to enhance cell affinity and wettability. The chitosan coated PLLA matrix induced increased osteoblast attachment as compared with intact PLLA surface. Overall results in this study demonstrated the usefulness of chitosan as drug releasing scaffolds and as modification tools for currently used biomaterials to enhance tissue regeneration efficacy. These results may expand the feasibility of combinative strategy of controlled local drug delivery concept and tissue engineered bone formation in reconstructive therapy in the field of periodontics, orthopedics and plastic surgery.

Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-beta1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly-l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.

BACKGROUND

It is known that platelet-derived growth factor (PDGF) receptors are expressed in hair follicle (HF) epithelium.

OBJECTIVE

The aim of the present study was to clarify the effects of PDGF-AA and -BB on the cyclic growth of HFs.

METHODS

PDGF-AA or -BB was injected into the dorsal skin of C3H mice during the second telogen phase once daily for five consecutive days, or PDGF-AA or -BB dissolved in hyaluronic acid was injected only once. In order to confirm the effects of different PDGF isoforms, anti-PDGF-AA antibody or anti-PDGF-BB antibody was injected just after each injection of PDGF-AA or -BB. In addition, anti-PDGF antibodies were injected into the skin of C3H mice during the second anagen phase once daily for 5 days. We studied expression of signaling molecules in the skin where anagen phase had been induced by PDGF injection by real-time RT-PCR.

RESULTS

Both PDGF-AA and -BB injection experiments immediately induced the anagen phase of the hair growth cycle at the injection sites. The induction of anagen was interfered by anti-PDGF antibody treatment. Real-time RT-PCR using extracted RNA from the PDGF injected sites of skin samples showed upregulated expression of HF differentiation-related key signaling molecules, Sonic hedgehog (Shh), Lef-1 and Wnt5a.

CONCLUSIONS

These results indicate that both PDGF-AA and -BB are involved in the induction and maintenance of the anagen phase in the mouse hair cycle. Local application of PDGF-AA and -BB might therefore prove to be an effective treatment option for alopecia associated with early catagen induction and elongated telogen phase.

Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230-270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.

The aim of our study was to further explore the use of anti-angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet-derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF-BB (B16/PDGF-BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size-matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF-BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF-BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of alpha-smooth muscle actin and PDGFRbeta-positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF-BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.

Apart from becaplermin (recombinant human platelet-derived growth factor homodimer of B chains, PDGF-BB), for the treatment of lower extremity diabetic ulcers, few agents are available for pharmacological stimulation of wound healing. We have compared the mechanism of action of the potential wound healing agent, PL 14736 (G E P P P G K P A D D A G L V), with that of PDGF-BB on granulation tissue formation following sponge implantation in the normoglycemic rat and in healing full-thickness excisional wounds in db/db genetically diabetic mice. Expression of the immediate response gene, early growth response gene-1 (egr-1) was studied in Caco-2 cells in vitro. While PDGF-BB and PL 14736 had similar selectivity for stimulation of granulation tissue in both sponge granuloma and in healing wounds in db/db mice, PL 14736 was more active in stimulating early collagen organization. It also stimulated expression of egr-1 and its repressor nerve growth factor 1-A binding protein-2 (nab2) in non-differentiated Caco-2 cells more rapidly than PDGF-BB. EGR-1 induces cytokine and growth factor generation and early extracellular matrix (collagen) formation, offering an explanation for the beneficial effects of PL 14736 on wound healing.

The binding of three radiolabeled isoforms of platelet-derived growth factor (PDGF), 125I-PDGF-AA, 125I-PDGF-AB, and 125I-PDGF-BB, is differentially affected by exposure of quiescent 3T3 cells to transforming growth factor-beta (TGF-beta). By 24 h after exposure to TGF-beta, binding of 125I-PDGF-AA and 125I-PDGF-AB is almost completely lost, whereas binding of 125I-PDGF-BB is reduced by only 40%. The loss of PDGF-binding sites caused by TGF-beta is time- and concentration-dependent and reflects a change in the pattern of expression of receptor subunits; the number of alpha-subunits decreases, and the number of beta-subunits increases. The loss of binding sites for PDGF-AA is accompanied by a decreased mitogenic response to PDGF-AA but not to PDGF-AB or PDGF-BB. These results suggest that TGF-beta may differentially regulate the expression of PDGF-binding sites and the mitogenic responsiveness toward the three PDGF isoforms. TGF-beta did not stimulate synthesis of PDGF A-chain mRNA or PDGF-AA protein, and PDGF-AA receptors could not be restored by the presence of suramin, suggesting that the loss of binding sites may result from direct effects on receptor expression rather than autocrine down-regulation by PDGF-AA.

Endogenous and transplanted neural stem cells (NSC) are highly migratory and display a unique tropism for areas of neuro-pathology. However, signals controlling NSC motility in health and disease are still ill-defined. NSC appear to be intimately associated with the cerebral vasculature and angiogenesis is a hallmark of many neurological disorders. This has led us to investigate the influence of quiescent and angiogenically active human endothelial cells on human NSC migration. In vivo we observed frequent perivascular accumulation of human NSC in the proximity of cerebral microvessels upon induction of angiogenesis by cerebral infusion of vascular endothelial growth factor (VEGF) into the murine brain. We analyzed the in vitro effects of conditioned media from human endothelial cells before and after angiogenic stimulation with VEGF on the migration of human NSC in vitro. Non-stimulated endothelial cells induced a moderate chemotactic migration that was significantly enhanced after angiogenic activation by VEGF. In order to identify cytokines that may function as stimulators of NSC chemotaxis, we screened endothelial cell-conditioned media for the expression of 120 different cytokines. We identified PDGF-BB, RANTES, I-TAC, NAP-2, GROalpha, Ang-2, and M-CSF as endothelial cell-released chemoattractants for human NSC in vitro. VEGF-stimulated cerebral microvascular endothelial cells secreted higher levels of Ang-2 and GROalpha, which in part were responsible for the enhanced chemoattraction of NSC. Our findings support the hypothesis that the angiogenically active microvasculature modulates the local guidance of NSC through endothelial cell-derived chemoattractants.

Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast-like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor-like cell line MC3T3-E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP-4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three-step chromatography by heparin affinity, anion exchange, and reversed-phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet-derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti-PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.

BACKGROUND

Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.

RESULTS

We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway. Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1 alpha, MIP-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.

CONCLUSIONS

The pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.

OBJECTIVE

Nitrovasodilators have been proposed for the treatment of portal hypertension alone or in combination with beta-blockers. In addition to their vasodilatory properties, nitric oxide (NO) donors may exert direct antifibrogenic properties. We evaluated the effect of nitroglycerin (NTG) and S-nitroso-N-acetyl penicillamine (SNAP) on the mitogenic and chemotactic properties of platelet-derived growth factor (PDGF)-BB and the modulation of the relative intracellular signaling pathways in fully activated human hepatic stellate cells (HSCs), a cell type that plays an active role in liver fibrogenesis and portal hypertension.

RESULTS

Both NTG and SNAP induced a dose-dependent decrease in PDGF-induced DNA synthesis and cell migration, which was associated with a decrease in PDGF-induced intracellular Ca(2+) increase and extracellular signal-regulated kinase (ERK) activity. These effects were not related to activation of the classic soluble guanylate cyclase (sGC)/guanosine 3',5'-cyclic monophosphate pathway; accordingly, Western blot analysis of HSC lysates revealed the absence of the alpha(1)beta(1) ubiquitous subunits of sGC, whereas they were detectable in quiescent HSCs, freshly isolated from normal human liver. Conversely, both NTG and SNAP induced a more than 10-20-fold increase in prostaglandin E(2) in cell supernatants within 1 minute, associated with an increase in intracellular adenosine 3',5'-cyclic monophosphate levels. Accordingly, the inhibitory effects of NO donors on PDGF action and signaling were eliminated after preincubation with ibuprofen.

CONCLUSIONS

These results suggest that NO donors may exert a direct antifibrogenic action by inhibiting proliferation, motility, and contractility of HSCs in addition to a reduction of fibrillar extracellular matrix accumulation.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.

3T3 L1-cells, which undergo adipose conversion in vitro, possess a stimulus-sensitive H2O2-generating system in their plasma membrane, and its properties are virtually identical with those of the insulin-sensitive human fat-cell oxidase [Krieger-Brauer and Kather (1992) J. Clin. Invest. 89, 1006-1013]. Insulin and insulin-like growth factor I were found to be active stimulators of NADPH-dependent H2O2 generation. Surprisingly, the acidic (a) and basic (b) isoforms of fibroblast growth factor (FGF) as well as the AA and BB homodimers of platelet-derived growth factor (PDGF) had antagonistic effects on NADPH-dependent H2O2 generation in plasma membranes which were parallelled by corresponding changes in H2O2 accumulation in intact cells. bFGF and PDGF BB (which inhibit NADPH-dependent H2O2 generation) prevented the adipose conversion of 3T3 L1-preadipocytes, and this effect could be reversed by exogenously supplied H2O2. Conversely, aFGF and PDGF AA, which stimulated H2O2 generation, accelerated adipocyte conversion in the presence of insulin and were adipogenic in themselves. Consistently, expression of the adipocyte phenotype induced by insulin, dexamethasone and isobutylmethylxanthine was enhanced in the presence of exogenous hypoxanthine/xanthine oxidase, whereas antioxidants, such as N-acetylcysteine or ascorbate, suppressed the process of differentiation. It is concluded that the H2O2 produced in response to hormones and cytokines may contribute to the development and maintenance of the differentiated state.

Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-beta receptor (PDGFbeta-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFbeta-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFbeta-R via the binding of tyrosine-phosphorylated Shc to PDGFbeta-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc.PDGFbeta-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFbeta-R by AngII (measured by PDGFbeta-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFbeta-R phosphorylation includes differences in the time course of PDGFbeta-R phosphorylation, differing effects of inhibitors of the endogenous PDGFbeta-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFbeta-R was directly immunoprecipitated (PDGFbeta-R-antibody) versus coimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc.PDGFbeta-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand.

BACKGROUND

The induction of angiogenesis after stroke may enhance neurorestorative processes. Our aim was to examine the endogenous angiogenesis balance and their association with long-term clinical outcome in ischemic stroke patients.

METHODS

A total of 109 stroke subjects were included in the study. Firstly, plasma samples were obtained from control subjects (n = 26) and tPA-treated stroke patients (n = 29) at baseline (within 3h of symptoms onset), 1, 2, 12, 24h after tPA treatment, at discharge and 3 months after the ischemic event. Angiogenic promoters (PDGF-AA, PDGF-BB, HGF, FGF, KGF, HB-EGF, TPO, VEGF, VEGFR-1, VEGFR-2 and SDF-1α) and inhibitors (endostatin, angiostatin, thrombospondin-1 and thrombospondin-2) were analyzed by Searchlight(®) technology or ELISA. Additionally, baseline and 24h endostatin plasma level was determined in a new set of stroke patients (n = 80). Clinical parameters (NIHSS, mRS, mortality and hemorrhagic transformation events) were assessed to evaluate outcome.

RESULTS

Baseline PDGF-BB, endostatin and thrombospondin-2 levels were higher in stroke patients than in controls (p < 0.05). A pro-angiogenic balance was associated with lower NIHSS scores and less intracranial hemorrhagic complications. Interestingly, a high baseline endostatin level was associated to long-term functional dependency (mRS>> 2; p = 0.004). Finally, a baseline endostatin cut-off point of 184 ng/mL was an independent predictor of functional dependency at three months in the multiple logistic regression with an odds ratio of 8.9 (95% CI: 2.7-28.8; p = 0.0002).

CONCLUSIONS

Our results indicate that an early pro-angiogenic balance is associated with mild short-term neurological deficit, while an acute anti-angiogenesis status determined by high endostatin plasma level predicts a worse long-term functional outcome.