The migration of arterial vascular smooth muscle cells (VSMC) is thought to play a central role in atherogenesis and restenosis. The migration of several other cell types, including monocytes, T-lymphocytes and endothelial cells is also involved in the development of the mature atherosclerotic lesion. Several defined growth factors, cytokines and extracellular matrix components which are released at the sites of lesions have been implicated in the regulation of migration of VSMC and other lesion-associated cells. Platelet-derived growth factor BB-homodimer of PDGF (PDGF-BB) is strongly implicated in neo-intima formation in vivo and is the most potent known chemoattractant for VSMC in vitro. Dynamic interactions between cell surface adhesive receptors (integrins) for ECM components, organisation of the actin cytoskeleton and the turnover of focal adhesions are all key processes in cell locomotion and migration. The signal transduction pathways which mediate the chemotactic effects of PDGF-BB and other migration factors on VSMC are unknown, but several classes of cellular components are implicated including components associated with focal adhesions, small GTP-binding proteins of the rho family, and certain substrates of the PDGF beta-receptor. Tyrosine phosphorylation of the novel focal adhesion-associated protein tyrosine kinase, p125 focal adhesion kinase (p125FAK), is regulated by integrins and by several factors which alter actin cytoskeletal organisation. Recent findings suggest that tyrosine phosphorylation of p125FAK and other focal adhesion-associated proteins may be implicated in the chemotactic response of VSMC to PDGF-BB. The migratory response to PDGF-BB may be dependent on both ligand isoform bio-availability and on receptor-isotype expression as well as on down-stream signalling events. Ultimately, cell migration in vivo will be determined by a complex array of diverse extracellular molecules organised in intercellular paracrine/autocrine networks as well as multiple interacting intracellular signal transduction pathways.

OBJECTIVE

To identify the in vitro characteristics and functional properties of fibrocytes and investigate the in vivo mechanism of action of fibrocytes injection in accelerating the cutaneous healing process in diabetic mice.

BACKGROUND

Fibrocytes are hematopoietic derived stem cells that may have a role in tissue repair, perhaps as the precursors of fibroblast- or myofibroblast-like cells.

METHODS

In vitro, the time-dependent phenotypic expression of peripheral blood (PB) fibrocytes was stained with anti-CD11b, anti-CD45, anti-Col-I, and anti-α-SMA antibodies. The functional properties of fibrocytes and dermal fibroblasts were tested by using reverse-transcriptase polymerase chain reaction. In vivo, full thickness wounds in diabetic mice were treated either with fibrocytes, dermal fibroblasts, or phosphate buffered saline (PBS) through tail vein injection. Wound healing kinetics, including wound contraction, re-epithelialization, and microscopic metrics such as cell proliferation, angiogenesis, and granulation growth were investigated. Expression of proinflammatory factors, profibrotic factors, growth factors, and extracellular matrix components were measured in wound tissues.

RESULTS

Fibrocytes gradually lose their hematopoietic cell markers and increase mesenchymal cell markers during differentiation in vitro. Fibrocytes stimulate wound healing by dermal cell proliferation, keratinocyte proliferation with re-epithelialization, and angiogenesis compared with dermal fibroblast and PBS treated wounds. Expression of angiogenesis markers (VEGF and b-FGF), growth factors (TGF-β, PDGF-A, and FGF-7), chemokines (MCP-1 and MIP-1α), and extracellular matrix (collagen-I and α-SMA) were upregulated in fibrocyte-treated wounds.

CONCLUSIONS

Peripheral blood fibrocytes can accelerate wound healing by stimulating cell proliferation, re-epithelialization, and angiogenesis in a diabetic mice experimental model. The application of fibrocytes may represent a potential clinical solution for the treatment of chronic wounds across all fields of surgery.

Tissue engineering of small-diameter blood vessels is still challenging because of restenosis and burst. To prevent thrombosis, rapid endothelialization along the lumen of grafts is intended, followed by proliferation of vascular smooth muscle cells (VSMCs) around the exterior for compliance. To this goal, two modified coaxial electrospinning techniques were developed to encapsulate vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGF), respectively, to regulate proliferation of vascular endothelial cells (VECs) and VSMCs. Release profiles, in vitro cell proliferation and in vivo implantation of double-layered electrospun membranes were investigated, and what made it special was the electrospun membranes were composed of chitosan hydrogel/poly(ethylene glycol)-b-poly(L-lactide-co-caprolactone) (PELCL) electrospun membrane loaded with VEGF as the inner layer and emulsion/PELCL electrospun membrane-loaded PDGF as the outer. It was found that dual-release of VEGF and PDGF could accelerate VEC proliferation in the first 6 days, and modulate slow VSMC proliferation in the initial 3 days whereas generate rapid proliferation after day 6, which is of great benefit to blood vessel regeneration. Four weeks of in vivo replacement of rabbit carotid artery demonstrated that VECs and VSMCs developed on the lumen and exterior of vascular grafts, respectively, and no thrombus or burst appeared. It was concluded that dual-delivery of VEGF and PDGF by the modified electrospun membranes could facilitate revascularization.

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45-60 years were excluded to help define "young" and "old" groups. Thirty-nine cases of sporadic EOCRC (patients ≤ 45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.

The blood-brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte-EC interaction during BBB development, the molecular mechanisms coordinating the pericyte-EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of <em>PDGF</em> receptor-<em>β</em> to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-<em>β</em>1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB.

Deposition of type III collagen protein is increased when cultured rat mesangial cells are cultured in media containing high glucose concentrations. Possible mechanisms for this effect include the production of growth factors, such as PDGF and TGF-beta, and the formation of abnormal glucose-protein adducts called advanced glycosylation end products (AGEs). In our studies, neutralizing antibodies to PDGF and TGF-beta prevented increased type III collagen deposition by mesangial cells exposed either to high glucose media or to low glucose media containing AGEs. Daily addition of PDGF or TGF-beta stimulated type III collagen production. However, while co-incubation with the TGF-beta Ab prevented PDGF-stimulated type III collagen production, the PDGF Ab did not prevent TGF-beta-stimulated type III collagen production. Daily addition of PDGF or TGF-beta stimulated, while AGEs inhibited, mesangial cell proliferation after 96 hours. We propose that high extracellular glucose and AGEs stimulate type III collagen production by pathways that involve the intermediate formation of PDGF and TGF-beta by mesangial cells. PDGF may increase type III collagen production by stimulating the intermediate production of TGF-beta. Exposure to high glucose, AGEs, or TGF-beta also leads to impaired mesangial cell proliferation. The autocrine effects of TGF-beta and PDGF play important roles in the effects of high extracellular glucose and AGEs on cultured mesangial cells.

Thrombin activates phosphoinositide 3-kinase (PI 3-kinase) in platelets via a mechanism involving G-proteins, possibly of both the heterotrimeric and the low molecular weight families. We have investigated the regulation of PI 3-kinase present in platelet cytosol, and we show that this activity can be stimulated by a mixed preparation of G-protein beta gamma-subunits. This stimulation is reversed by preincubation of the beta gamma-subunits with GDP-liganded alpha-subunits. The beta gamma-stimulated activity is inhibited by wortmannin, a recently identified inhibitor of PI 3-kinase in other systems. In addition, the activity associates with PDGF receptor phosphotyrosyl peptide and monoclonal antibody designed to interact with the p85 subunit of PI 3-kinase. We suggest that this beta gamma-sensitive activity is related to previously identified forms of PI 3-kinase.

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.

The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly(129)->>Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.

PURPOSE Dasatinib is an orally available tyrosine kinase inhibitor with low nanomolar activity against SRC family kinases, BCR-ABL, c-KIT, EPHA2, and the PDGF-β receptor. Dasatinib was found to have selective activity in several tumor models in the Pediatric Preclinical Testing Program. PATIENTS AND METHODS A phase I study of dasatinib in pediatric patients with refractory solid tumors or imatinib-refractory, Philadelphia chromosome-positive leukemia was performed. Dose levels of 50, 65, 85, and 110 mg/m²/dose, administered orally twice daily for 28 days, with courses repeated without interruption, were studied. Pharmacokinetic studies were performed with the initial dose.

RESULTS

A total of 39 patients (solid tumors, n = 28; chronic myeloid leukemia [CML], n = 9; acute lymphoblastic leukemia, n = 2) were enrolled. No dose-limiting toxicities (DLTs) were observed at the 50, 65, and 85 mg/m² dose levels. At 110 mg/m², two of six patients experienced DLT including grade 2 diarrhea and headache. In children with leukemia, grade 4 hypokalemia (50 mg/m²), grade 3 diarrhea (85 mg/m²), and grade 2 creatinine elevation (50 mg/m²) were observed. DLT in later courses included pleural effusions, hemangiomatosis, and GI hemorrhage. There were three complete cytogenetic responses, three partial cytogenetic responses, and two partial/minimal cytogenetic responses observed in evaluable patients with CML. CONCLUSION Overall, drug disposition and tolerability of dasatinib were similar to those observed in adult patients.

The ubiquitous Fer protein-tyrosine kinase has been proposed to regulate diverse processes such as cell growth, cell adhesion, and neurite outgrowth. To gain insight into the biological function of Fer, we have targeted the fer locus with a kinase-inactivating missense mutation (fer(D743R)). Mice homozygous for this mutation develop normally, have no overt phenotypic differences from wild-type mice, and are fertile. Since these mice lack both Fer and the testis-specific FerT kinase activities, these proteins are clearly not essential for development and survival. No differences were observed in overall cellularity of bone marrow, spleen, or thymus in the absence of Fer activity. While most platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation was unchanged in fer(D743R) homozygous embryonic fibroblasts, cortactin phosphorylation was reduced. However, Fer kinase activity was not required for PDGF-induced Stat3, p120(ctn), or epidermal growth factor (EGF)-induced beta-catenin phosphorylation. Also, no defects were observed in changes to the actin cytoskeleton, adherens junctions, or focal adhesions in PDGF- or EGF-stimulated fer(D743R) homozygous embryonic fibroblasts. Therefore, Fer likely serves a redundant role in regulating cell growth, cell adhesion, retinal development, and spermatogenesis but is required for efficient phosphorylation of cortactin.

Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.

Acidic and basic fibroblast growth factors (aFGF, bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor type beta (TGF-beta) are well-characterized growth regulators. Though there are reports of the effects of some of these factors on vascular cells, variability in the purity of growth factor preparations and differing cell culture conditions make comparison among the results difficult. Thus, a study employing homogeneous preparations of recombinant growth factors on well-characterized cell populations was conducted. Each of the factors was tested for its effect on the proliferation of cells derived from large and small blood vessels: aortic and capillary endothelial cells (EC), smooth muscle cells (SMC) and pericytes. Of the five growth factors only bFGF stimulated the proliferation of both large vessel and microvessel EC; aFGF was mitogenic for microvessel but not large vessel EC. Neither PDGF (AA, BB or AB) nor EGF had any effect on EC growth. TGF-beta was a potent inhibitor of both aortic and capillary EC proliferation with half maximal inhibition at concentrations as low as 0.05 and 0.25 ng/ml for microvessel and aortic EC, respectively. The FGFs were potent mitogens for the mural cells, SMC and pericytes. Likewise, PDGF was stimulatory for SMC and pericytes. The BB homodimer yielded the greatest degree of growth stimulation for both pericytes and SMC. In the case of SMC the AA homodimer stimulated small but significant growth and the effect of AB was intermediate to that of the homodimeric forms. On the other hand, neither the AA homodimer nor the heterodimer stimulated any significant increase in pericyte number. EGF was moderately mitogenic for both types of mural cells, reaching maximum stimulation at 100 pg/ml of EGF. TGF-beta was inhibitory for SMC but not for pericyte proliferation. These data indicate both quantitative and qualitative differences between the responses of large and small vessel EC to the various growth factors and distinct differences in the responsiveness of SMC and pericytes to PDGF and TGF-beta.

BACKGROUND

Connective tissue growth factor (CTGF) is a profibrotic growth factor, which is upregulated in wound healing and renal fibrosis, including anti-Thy-1.1 nephritis. The kinetics of CTGF mRNA expression in anti-Thy-1.1 nephritis suggested that CTGF regulation might contribute to glomerular response to injury downstream of transforming growth factor-beta (TGFbeta). In anti-Thy-1.1 nephritis the initial damage is followed by mesangial repair and limited sclerosis, which involves mesangial cell (MC) activation (alpha-smooth-muscle actin (alphaSMA) expression), proliferation, migration, and extracellular matrix production. The present in vitro study addresses the possible role of CTGF in these different aspects of mesangial response to injury, and how CTGF activity might relate to effects of TGFbeta and platelet-derived growth factor-BB (PDGF-BB).

RESULTS

Immunostaining and ELISA showed that alphaSMA expression and transformation of MC into myofibroblast-like cells was induced by TGFbeta, but not affected by PDGF-BB, CTGF, or neutralizing anti-CTGF antibodies. [(3)H]thymidine incorporation and Ki67 staining demonstrated that, unlike PDGF-BB, neither CTGF nor TGFbeta induced the proliferation of MC. In contrast, both CTGF and TGFbeta induced MC migration, as evidenced by approximation of wound edges in scrape-wounded, non-proliferating rat MC monolayers. In addition, fibronectin expression was upregulated by both CTGF and TGFbeta, as measured by dot-blot analysis. Anti-CTGF completely blocked the effect of added CTGF. Moreover, anti-CTGF significantly reduced TGFbeta-induced increase in fibronectin.

CONCLUSIONS

It thus appears that CTGF is specifically involved in a subset of the adaptive changes of MC involved in mesangial repair and sclerosis, which makes it an interesting candidate target for future intervention strategies.

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the beta 1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-beta, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the alpha 2 beta 1 and, to a lesser extent, the alpha 1 beta 1 integrin complexes. The present studies were undertaken to determine the role of the alpha 1 integrin chain, a collagen/laminin receptor, in collagen gel contraction by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the alpha 1 beta 1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the alpha 1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the alpha 1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the alpha 1 beta 1 integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.

The purpose of this study was to determine whether changes in the expression of platelet-derived growth factor (PDGF) and its receptors were associated with prostate cancer. Peroxidase-anti-peroxidase-immunoperoxidase labeling was used to detect the A and B chains and alpha and beta receptors of PDGF in 5 benign prostatic hyperplasias and 13 human prostate adenocarcinomas (Gleason grades 2 to 9). In all 5 benign prostatic hyperplasias, none of the PDGF antibodies (even at high titers, 1:50 dilutions) labeled the epithelial or the stromal cells. In adenocarcinomas, the A chain and alpha receptor antibodies labeled both epithelial and stromal cells. The intensity of the staining was relatively high in low Gleason grade (<6) and low in high Gleason grade (>7) tissue; however, the B chain and beta receptor antibodies failed to label either epithelial or stromal cells in any of the 13 adenocarcinomas. The data suggest that PDGF A and alpha receptor genes may be preferentially turned on in epithelial and stromal prostate tumor cells.

Human atherosclerotic plaques are heterogeneous tissues containing a number of different cell types, including macrophages, smooth muscle, endothelial and other undefined mesenchymal-appearing cells. Significant numbers of macrophages are found in human atherosclerotic plaques and have been postulated to be a major source of growth factor production during atherogenesis. In vitro evidence suggested that macrophages synthesize PDGF and might therefore contribute to the growth of the vessel wall in atherosclerosis. However, examination of PDGF synthesis in human atheroma by in situ hybridization revealed that while smooth muscle, mesenchymal, and endothelial cells synthesize this growth factor macrophages did not. Our inability to detect PDGF mRNA in macrophages was not due to any problems with hybridization to this cell type. In situ hybridization studies on human atherosclerotic plaques have demonstrated that plaque macrophages contain many different mRNAs other than PDGF including tissue factor, factor XIII, apoprotein E, transforming growth factor beta, and tumor necrosis factor. Recent studies have indicated that macrophages may be a major source as well of another group of inflammatory cytokines which are members of the RANTES/SIS cytokine family. In situ hybridization studies on human carotid endarterectomy specimens using probes specific for the inflammatory cytokines RANTES, LD78, HIMAP, and MCP-1 revealed numerous cells containing the mRNAs encoding for these proteins (5%, 13%, 8%, and 16% of plaque cells respectively). This is in contrast to generally low level expression found in normal human arteries (< 1% of normal medial cells contain these mRNAs). Cells expressing these cytokines were often found associated with inflammatory zones in human atherosclerotic plaques. Serial section immunohistochemistry suggests that macrophages and/or T cells may synthesize these proteins. In addition to localization to macrophages MCP-1 expression was also detected in smooth muscle cells and mesenchymal-appearing cells with many of the morphological characteristics of cells previously seen to express PDGF. In vitro evidence suggests that these proteins may be chemotactic to monocytes and lymphocytes. The finding of increased expression of these mRNAs in human atheroma suggests they may play a role in monocyte trafficking into the atherosclerotic plaque.

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.

The wild-type and two mitogenic-defective mutants of the type beta receptor for platelet-derived growth factor (PDGF) were expressed in Chinese hamster ovary cells. In the first mutant, delta Ki, 82 of 104 amino acids in the kinase insert region were deleted. This mutant was recently reported to be defective in mediating DNA synthesis. In the second mutant, Y825F, tyrosine 825 was converted to phenylalanine by a point mutation. We report here that this mutant is also defective in mediating PDGF-stimulated DNA synthesis. Both mutants were capable of eliciting many of the early responses to PDGF, including receptor autophosphorylation. However, neither mutant was capable of undergoing PDGF-stimulated change in receptor conformation or of phosphorylating exogenous substrate in an in vitro assay. These data suggest that changes in receptor conformation and efficient utilization of specific tyrosine kinase substrates are important for the stimulation of cell proliferation of PDGF and that phosphorylation of tyrosine 825 may be involved in signal transduction.

Oxidative stress plays an important role in the pathogenesis of Alzheimer's disease. To determine which mechanisms cause the origin of oxidative damage, we analyzed enzymatic antioxidant defense (Cu/Zn-superoxide dismutase Cu/Zn-SOD, glutathione peroxidase GPx and glutathione reductase GR) and lipid peroxidation products malondialdehyde MDA and 4-hydroxynonenal HNE in two different APP transgenic mouse models at 3-4 and 12-15 months of age. No changes in any parameter were observed in brains from PDGF-APP695(SDL) mice, which have low levels of Abeta and no plaque load. In contrast, Thy1-APP751(SL) mice show high Abeta accumulation with aging and plaques from an age of 6 months. In brains of these mice, HNE levels were increased at 3 months (female transgenic mice) and at 12 months (both gender), that is, before and after plaque deposition, and the activity of Cu/Zn-SOD was reduced. Interestingly, beta-amyloidogenic cleavage of APP was increased in female Thy1-APP751(SL) mice, which also showed increased HNE levels with simultaneously reduced Cu/Zn-SOD activity earlier than male Thy1-APP751(SL) mice. Our results demonstrate that impaired Cu/Zn-SOD activity contributes to oxidative damage in Thy1-APP751(SL) transgenic mice, and these findings are closely linked to increased beta-amyloidogenic cleavage of APP.

The growth factor PDGF controls the development of glioblastoma (GBM), but its contribution to the function of GBM stem-like cells (GSC) has been little studied. Here, we report that the transcription factor FoxM1 promotes PDGFA-STAT3 signaling to drive GSC self-renewal and tumorigenicity. In GBM, we found a positive correlation between expression of FoxM1 and PDGF-A. In GSC and mouse neural stem cells, FoxM1 bound to the PDGF-A promoter to upregulate PDGF-A expression, acting to maintain the stem-like qualities of GSC in part through this mechanism. Analysis of the human cancer genomic database The Cancer Genome Atlas revealed that GBM expresses higher levels of STAT3, a PDGF-A effector signaling molecule, as compared with normal brain. FoxM1 regulated STAT3 transcription through interactions with the β-catenin/TCF4 complex. FoxM1 deficiency inhibited PDGF-A and STAT3 expression in neural stem cells and GSC, abolishing their stem-like and tumorigenic properties. Further mechanistic investigations defined a FoxM1-PDGFA-STAT3 feed-forward pathway that was sufficient to confer stem-like properties to glioma cells. Collectively, our findings showed how FoxM1 activates expression of PDGF-A and STAT3 in a pathway required to maintain the self-renewal and tumorigenicity of glioma stem-like cells.

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml(-1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml(-1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml(-1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, micro-computed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo.

Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P(1)/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a G(i)-dependent manner, and activated ERK and p38 MAPK in G(i)- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn(17)Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SBPDGF mRNA and protein up-regulation, indicating the involvement of G(i)-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on G(i), Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P(1)-triggered, G(i)-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells.

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.