Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1(+)) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1(+) progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6-90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1(+) progenitors with antibodies against integrin alpha(1), alpha(v), and beta(1), but not beta(3), inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor-beta significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (alpha(1), beta(1), and alpha(v))-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor-beta signaling pathways are involved in SMC differentiation.

Although fracture healing is a well-optimized biological process that leads to healing, approximately 10-20% of fractures result in impaired or delayed healing and these fractures may benefit from the use of biotechnologies to enhance skeletal repair. Peptide signaling molecules such as the bone morphogenetic proteins have been shown to stimulate the healing of fresh fractures, nonunions, and spinal fusions and side effects from their use appear to be minimal. Other growth factors currently being studied for local application include growth and differentiation factor-5 (GDF-5), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and platelet-derived growth factor (PDGF). Molecules such as prostaglandin E receptor agonists and the thrombin-related peptide, TP508, have shown promise in animal models of fracture repair. Gene therapy using various growth factors or combinations of factors might also aid in fracture repair, particularly as new methods for delivery that do not require viral vectors are developed. Systemic therapy with agents such as parathyroid hormone (PTH), growth hormone (GH), and the HMG-CoA reductase inhibitors are also under investigation. As these and other technologies are shown to be safe and effective, their use will become a part of the standard of care in managing skeletal injuries.

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.

The (beta)1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of (beta)1 and (beta)3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the (beta)1A splice variant of (beta)1 ((beta)1A-GD25) did not express (alpha)2(beta)1A and did not adhere to collagen. After transfection of (alpha)2 into (beta)1A-GD25 cells, the (alpha)2(beta)1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas (beta)1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to (alpha)v(beta)3 or a RGD-containing peptide, indicating that (alpha)v(beta)3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by (alpha)2(beta)1A-GD25 cells was not inhibited by antibodies to (alpha)v(beta)3 or RGD-containing peptide, but was inhibited by anti-(alpha)2 antibody. Flow cytometry demonstrated negligible expression of (alpha)v(beta)3 by (beta)1A-GD25 and (alpha)2(beta)1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by (alpha)2(beta)1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by (alpha)2(beta)1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the (beta)1-null GD25 cells and (beta)1A and (alpha)2(beta)1A transfectants demonstrate that (alpha)2(beta)1A and (alpha)v(beta)3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.

Signals that promote proliferation and migration of smooth muscle cells (SMC) have been implicated in pathologic growth of hollow organs. Members of the platelet-derived growth factor (PDGF) family, potent mitogens and motility factors for SMC, have been shown to signal through cholesterol-enriched lipid rafts. We recently demonstrated that PDGF-stimulated DNA synthesis in urinary tract SMC was dependent on the integrity of lipid rafts. Despite its known ability to rapidly alter discrete proteins within rafts, the effect of PDGF on overall raft protein composition is unknown. In this study, we employed isotope coded affinity tag (ICAT) analysis to evaluate PDGF-induced protein changes in lipid rafts of primary culture human SMC. Following acute (i.e., 15 min) exposure of SMC to PDGF, 23 proteins increased in rafts >20%. In contrast, raft localization of only three proteins increased after 12 h of PDGF treatment. Among the proteins that increased at 15 min were the glycophosphatidylinositol-anchored proteins Thy-1, 5'-nucleotidase, and CD55, the cytoskeletal proteins actin, actinin, tropomyosin-3 and -4, and the endocytosis-related proteins clathrin and beta-adaptin. In addition, eight Rho family members were localized to rafts by ICAT analysis. Collectively, these observations suggest a role for lipid rafts in regulation of PDGF-stimulated changes in the cytoskeleton.

Phosphorylation of the p53 tumor suppressor protein is a critical event in the up-regulation and activation of p53 during cellular stress. In this study, we characterized the signaling pathway linking oxidative stress to p53 through the platelet-derived growth factor beta (PDGF beta) receptor and the ataxia telangiectasia mutated (ATM) kinase. In response to H2O2, we observed phosphorylation of p53 specifically at serine 15, but not serine 9, 20, or 392. Phosphorylation of Ser-15 was correlated with enhanced induction and functional activation of p53 manifest as transcription of the p53 target p21CIP/WAF. We found that H2O2 induced phosphorylation of the PDGF beta receptor and increased ATM kinase activity, two events integral to p53 activation as either AG1433 (a PDGF beta receptor inhibitor) or caffeine (an ATM kinase inhibitor) inhibited Ser-15 phosphorylation. Similarly, p53 activation by H2O2 was inhibited by kinase-inactive forms of the PDGF beta receptor or ATM. Inhibition of ATM kinase had no effect on H2O2-induced PDGF beta receptor tyrosine phosphorylation, whereas PDGF beta receptor suppression with RNA interference impaired H2O2-induced ATM activation, indicating that ATM lies downstream to the PDGF beta receptor in this signaling cascade. Functionally, inhibition of the PDGF beta receptor abrogated the inhibition of cell proliferation, and promotion of apoptosis due to H2O2 treatment. Thus, these data link PDGF beta receptor transactivation to H2O2-induced p53 phosphorylation and suggest a functional role for growth factor receptors in modulation of p53 function.

Activation of receptor tyrosine kinases is thought to involve ligand-induced dimerization, which promotes receptor transphosphorylation and thereby increases the receptor's phosphotransferase activity. We used two platelet-derived growth factor beta-receptor (beta-PDGFR) mutants to identify events that are required for full engagement (autophosphorylation and activation of the kinase activity) of the beta-PDGFR kinase. The F79/81 receptor (Tyr to Phe substitution at 579 and 581 in the juxtamembrane domain of the receptor) was capable of only very modest ligand-dependent autophosphorylation and also failed to associate with numerous SH2 domain-containing proteins. Furthermore, stimulation with platelet-derived growth factor (PDGF) did not increase the kinase activity of the F79/81 mutant toward exogenous substrates. However, the F79/81 receptor had basal kinase activity and could be artificially stimulated by incubation with ATP. Because the low kinase activity of the F857 mutant (Tyr to Phe substitution at 857 in the putative activation loop) could not be increased by incubation with ATP, failure to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has low kinase activity. Surprisingly, the F857 mutant underwent efficient PDGF-dependent autophosphorylation. Thus the PDGF-dependent increase in the kinase activity of the receptor is not required for autophosphorylation. We conclude that full activation of the beta-PDGFR kinase requires at least two, apparently distinct events.

We used the technique of in situ hybridization to determine if cells expressing PDGF B-chain mRNA can be detected in a model of mesangial proliferative nephritis in the rat induced with antibody directed against the Thy 1 antigen present on the mesangial cell membrane. The method involved hybridization with a digoxigenin-labeled cRNA probe for the murine PDGF B-chain followed by detection with an anti-digoxigenin-alkaline phosphatase conjugate and subsequent colorimetric reaction. In normal rats (N = 4), the majority of glomeruli (74%) were negative for PDGF B-chain mRNA, whereas 65% of glomeruli from rats with mesangial proliferative nephritis (N = 4) had segmental or diffuse staining for PDGF B-chain mRNA in a mesangial pattern. The difference, as measured using a semiquantitative scale, was significant (mean scores 0.4 +/- 0.2 vs. 1.9 +/- 0.2; scale 0 to 3+; P less than 0.001). The increase in PDGF B-chain mRNA positive cells localized to areas of hypercellularity and was associated with a significant increase in cells positive for PDGF B-chain by immunostaining with a specific monoclonal antibody (0.8 +/- 0.1 vs. 1.7 +/- 0.4, scale 0 to 3+, normal vs. diseased rats, P less than 0.005). Complement depletion, which prevents the mesangial cell proliferation, also prevented the increase in cells expressing PDGF B-chain mRNA and protein. Thus, this method of in situ hybridization can successfully detect cells expressing PDGF mRNA in active glomerulonephritis, and may be useful for detecting cells expressing genes for other growth factors and cytokines in both human and experimental models of glomerular injury.

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.

According to the cancer stem cell (CSC)/cancer-initiating cell hypothesis, glioma development is driven by a subpopulation of cells with unique tumor-regenerating capacity. We have characterized sphere-cultured glioma-derived cancer-initiating cells (GICs) from experimental gliomas induced by platelet-derived growth factor-B (PDGF-B) in neonatal Gtv-a Arf(-/-) mice. We found that the GICs can maintain their stem cell-like characteristics in absence of exogenous epidermal growth factor and fibroblast growth factor 2 and that this culture condition was highly selective for tumor-initiating cells where as few as five GICs could induce secondary tumor formation after orthotopic transplantation. Addition of FBS to the medium caused the GICs to differentiate into cells coexpressing glial fibrillary acidic protein and Tuj1, and this differentiation process was reversible, suggesting that the GICs are highly plastic and able to adapt to different environments without losing their tumorigenic properties. On inhibition of virally transduced PDGF-B by small interfering RNA treatment, the GICs stopped proliferating, lost their self-renewal ability, and started to uniformly express CNPase, a marker of oligodendrocyte precursor cells and mature oligodendrocytes. Most importantly, PDGF-B depletion completely abrogated the tumor-initiating capacity of the GICs. Our findings suggest that interfering with PDGF-controlled differentiation could be a therapeutic avenue for patients diagnosed with the PDGF-driven proneural subtype of human glioblastoma.

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.

Platelet-derived growth factor receptors (PDGFRs) and their ligands, platelet-derived growth factors (PDGFs) play critical roles in mesenchymal cell migration and proliferation. In embryogenesis the PDGFR/PDGF system is essential for the correct development of the kidney, cardiovascular system, brain, lung and connective tissue. In adults, PDGFR/PDGF is important in wound healing, inflammation and angiogenesis. Abnormalities of PDGFR/PDGF are thought to contribute to a number of human diseases, and especially malignancy. Constitutive activation of the PDGFRalpha or PDGFRbeta receptor tyrosine kinases is seen in myeloid malignancies as a consequence of fusion to diverse partner genes, and activating mutations of PDGFRalpha are seen in gastrointestinal tumours (GISTs). Autocrine signalling as a consequence of PDGF-B overexpression is clearly implicated in the pathogenesis of dermatofibrosarcoma protruberans (DFSP) and overexpression of PDGFRs and/or their ligands has been described in many solid tumours. PDGFR signalling is inhibited by imatinib mesylate, and this compound has clear clinical activity in patients with myeloid malignancies, GIST and DFSP.

Endothelin (ET), a peptide originally isolated from the supernatants of cultured endothelial cells, exerts a wide variety of biological effects in different tissues. Endothelial-cell-synthesized ET-1 has been proposed to act in a paracrine manner on adjacent smooth muscle cells (SMC) in vivo, with effects that include both vascular reactivity (vasodilation/vasoconstriction) and mitogenesis. This study, by the use of immunocytochemically characterized SMC (rVSMC) isolated from the aortas of spontaneously hypertensive rats, has investigated a possible autocrine role for ET in regulation of the vasculature. Although quiescent cultures of rVSMC apparently did not constitutively express prepro ET-1mRNA, ET-specific transcripts could be induced by a variety of growth factors (transforming growth factor beta [TGF-beta]; platelet-derived growth factor-AA homodimer [PDGF-A chain]) and vasoactive hormones (angiotensin II [Ang II], arginine-vasopressin, and ET-1 itself). The kinetics for prepro ET-1mRNA induction in rVSMC were characteristically rapid in onset and transient. Down-regulation of protein kinase C by 48 h pretreatment of rVSMC with phorbol ester markedly reduced the subsequent ability of rVSMC to express ET-1 transcripts and secrete ET-1 peptide in response to Ang II. Inducible prepro ET-1mRNA expression was accompanied by a cycloheximide-inhibitable release of ET-1 peptide into the medium of rVSMC. ET-1 peptide was determined by both radioreceptor- and radioimmunoassay. Stimulated rVSMC accumulated ET-1 (approximately 200 pg.10(6) cells-1 x 4 h-1) at levels that attained biological relevance (approximately 10(-10) M). Sep-pak C18 extracts of medium from stimulated rVSMC elicited contraction of isolated endothelium-denuded rat mesenteric resistance vessels, and this response was characteristically protracted and difficult to "wash out." Synthetic (porcine) ET-1 promoted the expression of transcripts for PDGF-A chain, TGF-beta, and thrombospondin in quiescent rVSMC. Such effects of ET-1 on gene expression may be relevant to the mitogenic potential of ET-1 on VSMC. Our findings imply a role for ET-1 in the control of vascular function via both paracrine and autocrine regulatory mechanisms. The expression of prepro ET-1mRNA and peptide biosynthesis by rVSMC may have both short-term (e.g., vasoconstriction) and long-term (e.g., structural remodeling) consequences. A sustained loop of autocrine stimulation by ET-1 in SMC could contribute toward the pathogenesis of vasospasm and/or atherosclerosis.

Systemic sclerosis (SSc) is an autoimmune disorder with clinical manifestations resulting from immune activation, fibrosis development and damage of small blood vessels. Although there have been no established treatments for SSc, lots of new treatments targeting organ and pathogenesis are in the process of development. Transforming growth factor (TGF)-beta is a major cytokine involved in the pathogenesis of fibrosis in SSc. The blockade of cell surface molecules capable of activating latent TGF-beta, blockade of ligand by the pan-isoform-specific antibody, soluble TGF-beta receptors and a recombinant latency associated peptide, as well as inhibitors for ALK5 and Smad3 are the potential strategies to abolish the pathological activation of TGF-beta signaling in SSc fibroblasts. Besides TGF-beta, connective tissue growth factor (CTGF)/CCN2, platelet-derived growth factor (PDGF) and endothelin-1 are the candidates for the new therapeutic targets. As for immune dysfunction in SSc, i.v. immunoglobulin infusion, stem cell transplantation and B-cell depletion are potential new therapies under or awaiting a randomized, double-blind, placebo-controlled trial, although their efficacies are still controversial. Phosphodiesterase-5 inhibitors, endothelin receptor antagonists and inhibitors for serotonin signaling are the new therapeutic targets for Raynaud's phenomenon, digital ulceration and pulmonary arterial hypertension in SSc. Imatinib mesylate may be a novel new therapy for fibrosis and vasculopathy in SSc because it reverses the expression levels of Fli1, which is a transcription factor downregulated in SSc through an epigenetic mechanism and is likely to be involved in the development of fibrosis and vasculopathy in this disease. Potential therapeutic targets other than those described above are also reviewed.

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

An SH2 domain originally termed SH2-B had been identified as a direct cellular binding target of a number of mostly mitogenic receptors. The complete cellular protein, termed PSM, and respective sequence variants share additional Pro-rich and PH regions, as well as similarities with APS and Lnk. A role of these mediators has been implicated in signaling pathways found downstream of growth hormone receptor and receptor tyrosine kinases, including the insulin, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), nerve growth factor, hepatocyte growth factor, and fibroblast growth factor receptors. As a result of this report a total of four PSM/SH2-B sequence variants termed alpha, beta, gamma, and delta have now been identified in the mouse and have been compared with the available rat and human sequences. Variant differences are based on alternative splicing and define distinct last exons 7, 8, and 9 that result in reading frameshifts and unique carboxyl-terminal amino acid sequences. Variant sequences have been identified from cDNA libraries and directly by reverse transcription-polymerase chain reaction. Sequence analysis predicts four distinctly sized protein products that have been demonstrated after cDNA expression. All were found phosphorylated on tyrosine specifically in response to IGF-I and PDGF stimulation. cDNA expression of the four variants caused variant-dependent levels of stimulation of IGF-I- and PDGF-induced mitogenesis. The most pronounced increase in mitogenesis was consistently observed for the gamma variant followed by delta, alpha, and beta with decreasing responses. In contrast, the mitogenic response to epidermal growth factor consistently remained unaffected. The variants are expressed in most mouse tissues, typically, most strongly in pairs of alpha and delta or beta and gamma. Our findings implicate differential roles of the PSM/SH2-B splice variants in specific mitogenic signaling pathways.

TNF-alpha stimulates DNA synthesis and proliferation of cultured human fibroblasts. Maximal DNA synthesis in response to TNF-alpha occurs approximately 28 h after addition of TNF-alpha to quiescent fibroblasts, a delay of about 12 to 14 h as compared with DNA synthesis elicited by platelet-derived growth factor (PDGF). TNF-alpha induces PDGF A chain gene expression with a maximum at 4 h. DNA synthesis is abrogated in response to TNF-alpha by a goat anti-PDGF IgG but not by nonimmune goat IgG, suggesting induction of an autocrine PDGF-AA loop by TNF-alpha. The response to PDGF-AA requires the presence of PDGF receptor alpha-receptors. TNF-alpha does not significantly affect PDGF alpha-receptor mRNA or protein expression, nor does it alter the proliferative response to externally added PDGF-AA. In contrast, TNF-alpha reduces the levels of PDGF beta-receptor mRNA, protein expression, and cell proliferation in response to PDGF-BB. Thus, DNA synthesis in response to TNF-alpha depends upon autocrinely induced PDGF-AA. At the same time, TNF-alpha may alter the response to PDGF-BB from exogenous sources.

Mesenchymal stem cell (MSC) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/beta-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled-related protein-1 (sFRP1), a modulator of the Wnt/Fz pathway, could modify MSC capacities, endowing MSC to increase vessel maturation. In the engraftment model, we show that murine bone marrow-derived MSC induced a beneficial vascular effect through a direct cellular contribution to vascular cells. MSC quickly organized into primitive immature vessel tubes connected to host circulation; this organization preceded host endothelial cell (EC) and smooth muscle cell (SMC) recruitment to later form mature neovessel. MSC sustained neovessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neovessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased platelet-derived growth factor-BB (PDGF-BB) expression in MSC and enhanced beta-catenin-dependent cell-cell contacts between MSC themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neovessels and vessel maturation through a glycogen synthase kinase 3 beta (GSK3beta)-dependent pathway. sFRP1-overexpressing MSC compared with control MSC were well elongated and in a closer contact with the vascular wall, conditions required to achieve an organized mature vessel wall. We propose that genetically modifying MSC to overexpress sFRP1 may be potentially effective in promoting therapeutic angiogenesis/arteriogenesis processes. Disclosure of potential conflicts of interest is found at the end of this article.

OBJECTIVE

This study evaluates the temporal sequence and growth factor release from platelet-rich plasma (PRP) combined with different bone substitutes (BS), to identify an optimal substrate for extended growth factor retention.

METHODS

PRP was clotted with bovine thrombin or thrombin receptor activator peptide-6 (TRAP). In addition, PRP was clotted using Allogro (Ceramed, Lakewood, CO), BioGlass (Mo-Sci, Rolla, MN), or BioOss (Osteohealth, Shirley, NY). The effects of media exchange and BS on platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF beta) release were quantified via enzyme-linked immunosorbent assay.

RESULTS

At day 1, the thrombin group released 36% more PDGF than the TRAP group and 80% more than the BS groups. At 7 days, PDGF release was the greatest for the TRAP group. PDGF release was minimal for all groups at day 14, with BS groups retaining 60% more PDGF than thrombin clots. Similarly, the thrombin group released the greatest amount of TGF beta (81.4% of the total), whereas TRAP and BS groups released significantly less TGF beta at day 1. Compared with thrombin, TRAP retained 39.2% more TGF beta, whereas BS groups retained even greater levels (Allogro, 54.3%; BioOss, 45.8%; BioGlass, 67.0%). No significant difference in TGF beta release was observed among the substitutes after day 1. The BS groups continued to retain TGF beta after 14 days, whereas all TGF beta in the thrombin clots was depleted.

CONCLUSIONS

PRP preparation with thrombin results in a large, immediate release of growth factors that could be lost into the interstitium in vivo. TRAP-BS may prove more efficacious than thrombin in sustaining growth factor levels critical for the cascade of events leading to bone formation.

P311, also called PTZ17, was identified by suppressive subtraction hybridization as potentially involved in smooth muscle (SM) myogenesis. P311 is an 8-kDa protein with several PEST-like motifs found in neurons and muscle. P311 transfection into two fibroblast cell lines, NIH 3T3 and C3H10 T1/2, induced phenotypic changes consistent with myofibroblast transformation, including upregulation of SM alpha-actin and SM22, induction of FGF-2, VEGF, <em>PDGF</em>, and <em>PDGF</em> receptors, upregulation of integrins alpha3 and alpha5, and increased proliferation rate. The P311-mediated changes differed, however, from the well-characterized myofibroblast in that P311 inhibited TGF-<em>beta</em>1, TGF-<em>beta</em> receptor 2, and TGF-<em>beta</em>1-activating MMP-2 and MMP-9, with the resultant decrease in collagen 1 and 3 expression. The effect of P311 on collagen was overcome by exogenous TGF-<em>beta</em>1, indicating that the cells were responsive to TGF-<em>beta</em>1 paracrine stimulus. In support of a role for P311 in vivo, immunohistochemical examination of human wounds showed P311 only in myofibroblasts and their activated precursors. To our knowledge, these studies are the first to implicate P311 in myofibroblast transformation, to demonstrate that transformation may occur independently of TGF-<em>beta</em>1, and to suggest that P311 may prevent fibrosis.

OBJECTIVE

Bone marrow-derived human mesenchymal stem cells (hMSCs) are capable of localizing to gliomas after systemic delivery and can be used in glioma therapy. However, the mechanism underlying the tropism of hMSCs for gliomas remains unclear. In vitro studies suggest that platelet-derived growth factor BB (PDGF-BB) may mediate this tropism. However, a causal role of PDGF-BB has not been demonstrated in vivo. Therefore, we tested the hypothesis that PDGF-BB mediates the attraction of hMSCs to gliomas in vitro and in vivo.

METHODS

U87 or LN229 glioma cells were transfected with plasmids encoding human PDGF-B. Stable transfected clones that secreted large amounts of PDFG-BB and clones that produced low levels of PDGF were chosen. In vitro migration of hMSCs toward PDGF-B or conditioned media from high- and low-secreting PDGF-B tumor cells was assessed using Matrigel invasion assays. For in vivo localization studies, hMSCs were tracked by bioluminescence imaging (BLI) after transduction with an adenovirus containing luciferase cDNA. In other studies, hMSCs were labeled with green fluorescent protein (gfp) and analyzed for intratumoral localization by immunohistochemistry.

RESULTS

In vitro invasion assays showed that significantly more hMSCs migrated toward glioma cells engineered to secrete high levels of PDGF-BB compared with low-secreting gliomas. Anti-PDGF-BB-neutralizing antibody abrogated this increase in migration. Pretreatment of hMSCs with inhibitory antibodies against PDGF receptor-beta also reduced hMSC migration. To demonstrate that PDGF-BB mediates the localization of hMSCs in vivo, hMSCs-Ad-Luc were injected into the carotid artery of mice harboring orthotopic 7-day-old U87-PDGF-BB-high secreting or U87-PDGF-BB-low secreting xenografts and analyzed by BLI. Statistically significant increases in hMSCs were seen within PDGF-BB-high xenografts compared with PDGF-BB-low xenografts. To control for PDGF-BB-induced differences in tumor size and vascularity, gfp-labeled hMSCs were injected into the carotid arteries of animals harboring 4-day old PDGF-BB-high secreting xenografts or 7-day old PDGF-BB-low secreting xenografts. At these times tumors had similar size and vessel density. Statistically significant more hMSCs localized to PDGF-BB-high secreting xenografts compared with PDGF-BB-low secreting xenografts. Pretreatment of hMSCs with anti-PDGFR-beta-inhibitory antibodies decreased the localization of hMSCs in this intracranial model.

CONCLUSIONS

PDGF-BB increases the attraction of hMSCs for gliomas in vitro and in vivo, and this tropism is mediated via PDGF-beta receptors on hMSCs. These findings can be exploited for advancing hMSC treatment.

Previous investigators reported that epiretinal membranes isolated from patients with proliferative vitreoretinopathy (PVR) express various platelet-derived growth factor (PDGF) family members and PDGF receptors (PDGFRs) (Cui, J.Z., Chiu, A., Maberley, D., Ma, P., Samad, A., Matsubara, J.A., 2007. Stage specificity of novel growth factor expression during development of proliferative vitreoretinopathy. Eye 21, 200-208; Robbins, S.G., Mixon, R.N., Wilson, D.J., Hart, C.E., Robertson, J.E., Westra, I., Planck, S.R., Rosenbaum, J.T., 1994. Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases. Invest. Ophthalmol. Vis. Sci. 35(10), 3649-3663). Co-expression of ligand and receptor raises the possibility of an autocrine loop, which could be of importance in the pathogenesis of PVR. To begin to address this issue we determined whether the PDGFRs in epiretinal membranes isolated from PVR patient donors were activated. Indeed, immunohistochemical staining (using pan- and phospho-PDGFR antibodies) revealed that both PDGFR subunits were activated. Quantification of these data demonstrated that a greater percentage of cells expressed the PDGFR alpha subunit as compared with the beta subunit (44 +/- 13% versus 32 +/- 6.5%). Staining with phospho-PDGFR antibodies indicated that 36 +/- 10% of the PDGFR alpha subunits were activated, whereas only 16 +/- 5.5% of the PDGFR beta subunits were activated. Thus, a 2.25 fold greater percentage of the PDGFR alpha subunits was activated. Co-staining with diagnostic cell-type antibodies indicated that both retinal pigment epithelial and glial cells expressed activated PDGFR alpha subunits. These findings support the recent discovery that PDGF-C is the major vitreal isoform because PDGF-C is 3 times more likely to activate a PDGFR alpha subunit as compared with a PDGFR beta subunit. We conclude that PDGFRs are activated in epiretinal membranes of patients with PVR, and that the profile of active PDGFR subunits functionally supports the idea that PDGF-C is the predominant PDGF isoform present in the vitreous of patients with PVR. These findings identify PDGF-A, -AB and C as the best therapeutic targets within the PDGF family.

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

This review focuses on the roles of NADPH oxidase/NOX proteins in idiopathic pulmonary fibrosis (IPF) pathophysiology and in the signalling pathways involved in IPF. NOX proteins are membrane-associated multi-unit enzymes that catalyze the reduction of oxygen using NADPH as an electron donor. Recent studies indicate that NOX4 is induced in pulmonary fibroblasts in response to TGF-β. TGF-β or PDGF induce myofibroblast proliferation, differentiation, migration, contractility and extracellular matrix production, through NOX4 and reactive oxygen species dependent SMAD2/3 phosphorylation. NOX4 is increased in pulmonary fibroblasts from IPF patients and deletion of Nox4 in mice prevents bleomycin-induced pulmonary fibrosis. These data strongly suggest that targeting of NOX4 could be a step forward in the treatment of fibrotic lung diseases, by specifically targeting myofibroblasts, a major player in this disease.