Platelet-derived Growth factor-AA Research Articles

Neutrophil elastase induces cell proliferation and migration by the release of TGF-α, PDGF and VEGF in esophageal cell lines

Rapid regrowth or recurrent growth of occult cancer cells are often observed after esophagectomy or postoperative complications. In order to clarify the mechanism of such oncological circumstances, we focused on neutrophil elastase (NE), which degrades a broad spectrum of extracellular matrix and cell surface proteins. In the present study, we demonstrated that NE stimulated the growth of all of the five esophageal cell lines (TE-1, -7, -8, -12 and -13) by MTT assay and promoted cell invasion by cell migration assay. Pro-transforming growth factor-alpha (pro-TGF-alpha) from the cell membrane was released to the culture medium as a mature form after treatment with 5 microg/ml NE, and it reached the maximum level of 153% compared to the control values at 15 min of treatment in TE-13 cells. The phosphorylation of epidermal growth factor receptor (EGFR) rapidly occurs after treatment with NE and triggered the extracellular signal-regulated kinases 1 and 2 (ERK) signaling pathway. Moreover, NE induced release of platelet-derived growth factor-AA (PDGF-AA), PDGF-BB and vascular endothelial growth factor (VEGF) to 141.9, 227.7, and 171.6% of the control values, respectively. A specific NE inhibitor, sivelestat, significantly inhibited the NE-induced cell proliferation, cell invasion and subsequently inhibited the signal transduction pathway. Furthermore, sivelestat significantly inhibited NE-induced release of TGF-alpha, PDGF-AA, PDGF-BB and VEGF in the medium in TE-13 esophageal carcinoma cells. These results strongly indicate that NE released from activated neutrophils stimulates the growth and progression of esophageal cancer cells by releasing the growth factors on the cell surface and that sivelestat, a specific NE inhibitor, blocks these processes. Furthermore, we postulate that postoperative administration of sivelestat might be useful as a new molecular-targeting cancer therapy as well as for the treatment of postoperative respiratory complications.

Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies

Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.

PDGF and microvessel wall remodeling in adult rat lung: imaging PDGF-AA and PDGF-Rα molecules in progenitor smooth muscle cells developing in experimental pulmonary hypertension

Smooth muscle cells are mostly absent from the walls of microvessels in the adult lung but develop in large numbers as part of the pathology of human and experimental pulmonary hypertensions (PHs). We have previously shown, in an in vivo model of experimental PH, that mesenchymal (interstitial) fibroblasts and intermediate cells are the progenitors of these cells. Although smooth muscle cell development is a defining pathophysiological feature of human PH, little is known about the angiogenic signaling molecules responsible. Here, we report data for platelet-derived growth factor AA (PDGF-AA) and PDGF-Ralpha, two components of an important signaling pathway for fibroblast and myofibroblast proliferation and migration. Using antibodies linked to protein-A gold and high-resolution imaging techniques, we analyzed the expression of these molecules as smooth muscle cells developed from progenitor cell populations and in endothelial cells of the same microvessels. PDGF-AA was highly expressed by each cell type in control lung. As PH developed, the number of antigenic sites for PDGF-AA decreased with time. PDGF-Ralpha expression levels in the control lung were low, relative to the ligand, and fell in PH. These data show, for the first time, a marked phenotypic shift in expression levels of the PDGF-AA isoform and its receptor tyrosine kinase in the progenitor smooth muscle cells developing in the microvessels of the adult hypertensive lung.

Combination Therapy of Imatinib Mesylate and Interferon-α Demonstrates Minimal Activity and Significant Toxicity in Metastatic Renal Cell Carcinoma: Results of a Single-Institution Phase II Trial

Background Renal cell carcinoma (RCC) is characterized by increased expression of vascular endothelial growth factor and platelet-derived growth factor (PDGF)–β, both of which contribute to its angiogenic phenotype. Interferon-α (IFN-α) improves survival in patients with metastatic RCC, perhaps partly because of its antiangiogenic properties. Imatinib mesylate inhibits PDGF-mediated signal transduction and might thus have antiangiogenic activity as well. Patients and Methods Patients with metastatic RCC were treated with IFN-α (9 × 10 6 IU subcutaneously 3 times weekly) and oral imatinib mesylate (600 mg daily starting on day 8). Therapy was continuous, and response was evaluated at 8-week intervals using the Response Evaluation Criteria in Solid Tumors. Baseline plasma PDGF-AA, PDGF-AB, and PDGF-BB levels were obtained. Results Between January 2003 and January 2005, 17 patients were treated. One patient (6%) had a partial response, 4 (24%) had stable disease, 7 (41%) had progressive disease, and 5 (29%) were unevaluable because of early withdrawal secondary to toxicity. Median time to progression (TTP) using the Kaplan-Meier method was 8 weeks, and median overall survival was 17.8 months. Six patients (35%) withdrew from therapy because of toxicity, and 9 patients (53%) experienced ≥ 1 grade 3/4 toxicity. Platelet-derived growth factor AA, AB, and BB plasma levels did not correlate with TTP or overall survival. Conclusion Based on a response rate of only 6%, a median TTP of 2 months, and significant toxicities, further study of IFN-α in combination with imatinib mesylate is not recommended in patients with metastatic RCC.

The gene expression profile of PDGF-treated neural stem cells corresponds to partially differentiated neurons and glia

We have previously shown that platelet-derived growth factor AA (PDGF-AA) stimulates the expansion of neuronal progenitors from neural stem cells, but is unable to replace fibroblast-growth factor 2 (FGF-2) as a stem cell mitogen. In the present study, we compared gene expression in neural stem cells that were grown in the presence of FGF-2 and in cells cultured with PDGF-AA or in the absence of growth factor, which induces differentiation. The genetic program elicited by PDGF-AA (156 significantly regulated genes) was not unique, but an intermediate between the ones of FGF-2-cultured stem cells and differentiated cells. These observations are compatible with the hypothesis that PDGF-AA induces a partial differentiation of neural stem cells, which retain the ability to proliferate, rather than acting solely as an instructing agent for neuronal differentiation. Finally, the transcriptional signature of stem cells grown with FGF-2 included a large number of genes over-expressed in gliomas and a core set of conserved genes periodically expressed during the eukaryote cell cycle.

Release of platelet‐derived growth factors and proliferation of fibroblasts in the releasates from platelets stored in the liquid state at 22°C after stimulation with agonists

Fresh platelet (PLT)-rich plasma (PRP) treated with thrombin plus calcium chloride (CaCl(2)) is used to prepare a PLT gel to promote hemostasis and wound healing in a variety of surgical procedures. The effects of various agonists on stimulating the release of growth factors from liquid-preserved PLTs and the effects of the PLT releasate on the growth of fibroblasts in tissue culture were investigated. Plateletpheresis PLTs stored at 22 degrees C as high-yield PLTs for 3 to 6 days or outdated PLTs for 9 days were treated with agonists to assess release of platelet-derived growth factor (PDGF) AA, PDGF AB, PDGF BB, transforming growth factor-beta1 (TGF-beta1), and osteocalcin and the proliferation of fibroblasts treated with the PLT releasates in tissue culture. All treatments except for CaCl(2) alone and zeolite-CaCl(2) produced significant increases in PDGF AA compared to PRP. Thrombin-CaCl(2) produced significant increases in PDGF BB. Treatment by all the agonists produced similar increases in PDGF AB. TGF-beta1 and osteocalcin levels after treatment were similar to those in PRP. PRP releasate before and after stimulation with different agonists increased proliferation of fibroblasts in tissue culture. High-yield and outdated liquid-preserved PLTs released PDGF AA, AB, and BB but not TGF-beta1 or osteocalcin. The releasate from untreated PRP stimulated the proliferation of fibroblasts in tissue culture similar to the releasates from PRP treated with the different agonists. Further studies are needed to assess whether or not high-yield and outdated PLTs may be useful in wound healing.

Platelet-Derived Growth Factor-AA Is an Essential and Autocrine Regulator of Vascular Endothelial Growth Factor Expression in Non–Small Cell Lung Carcinomas

It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non-small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA-dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

Coexpression of Angiogenic Factors in Brain Arteriovenous Malformations

Brain arteriovenous malformations (BAVMs) are structurally unstable blood vessels that display an angiogenic phenotype, possibly maintained by concerted effects of key angiogenic factors. Therefore, we hypothesized that there are close correlations among key angiogenic factors in BAVMs and that levels of key angiogenic factors are associated with BAVM clinical characteristics that are linked with vascular instability. We measured the expression of angiopoietin-2 (Ang-2), matrix metalloproteinase-9, vascular endothelial growth factor (VEGF), and platelet-derived growth factor-AA and -BB by use of enzyme-linked immunosorbent assay in 27 BAVM surgical specimens. Tissues were also collected from 14 structurally normal brain specimens obtained during epilepsy surgery. Ang-2, VEGF, and matrix metalloproteinase-9 were highly expressed in BAVM samples (11.9 +/- 15.5 ng/mg protein, 137 +/- 102 pg/mg protein, and 379 +/- 455 pg/mg protein, respectively). Platelet-derived growth factor-BB was detectable in 7 of 21 BAVM samples. There were close correlations between Ang-2 and VEGF (R2 = 0.79). Higher Ang-2 and VEGF levels seemed to be associated with draining vein characteristics linked with BAVM hemorrhage but not with feeding artery pressure or BAVM size. In the structurally normal brain specimens, levels of Ang-2, VEGF, and matrix metalloproteinase-9 were low (0.8 +/- 2.3 ng/mg protein, 38 +/- 25 pg/mg protein, and 52 +/- 43 pg/mg protein, respectively). There were close correlations among angiogenic factors in BAVMs. Concerted effects of angiogenic factors may maintain the angiogenic phenotype in BAVMs and thereby determine the clinical course of BAVMs.

Growth factor-induced signaling of the pancreatic epithelium

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH2-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.

Inhibition of Src-like Kinases Reveals Akt-dependent and -independent Pathways in Insulin-like Growth Factor I-mediated Oligodendrocyte Progenitor Survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.

Platelet-derived growth factor and platelet profiles in childhood nephrotic syndrome

The aim of the study was to investigate (1) whether there are any changes in release of platelet-derived growth factor AA (PDGF AA) in children with nephrotic syndrome without clinical thromboembolic symptoms 2; (2) whether serum PDGF AA correlates with the platelet count (PLT) and platelet indices; (3) whether prednisone therapy affects the serum PDGF AA and the PLT; (4) whether PDGF AA is a useful predictor of disease activity. The study involved two groups of children: 33 with nephrotic syndrome (I) who were evaluated twice (A during relapse and B after 2 weeks of prednisone treatment) and 34 healthy children (II). The serum concentration of PDGF was measured by ELISA. In group I/A the PLT (P<0.01) and platelet distribution width (P<0.05) were elevated, the mean platelet volume (MPV) (P<0.05) was decreased and the plateletcrit (P>0.05) was normal. In group I/B, the PLT was decreased and MPV increased. The concentration of PDGF AA was still increased and correlated negatively with the albumin concentration. Hence in children with nephrotic syndrome an increase in PLT, a decrease in MPV, and a higher concentration of PDGF were observed. Treatment of nephrotic syndrome with prednisone for 2 weeks is not sufficient to normalize platelet parameters. Further studies are necessary to confirm the role of PDGF AA in the hypercoagulation state in children with nephrotic syndrome.

To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGF.

The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-beta1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone.

A potential oncogenic activity of platelet-derived growth factor d in prostate cancer progression.

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH(2)-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the beta-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer.

Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines

Objectives. We determined the expression of platelet-derived growth factor (PDGF), PDGF-receptor (PDGF-R), and phosphorylated PDGF-R (p-PDGF-R) on tumor cells and tumor-associated endothelial cells in clinical specimens of human ovarian carcinoma and human ovarian cancer cells growing in culture and in the peritoneal cavity of nude mice. Methods. Ten specimens of high-grade serous ovarian carcinoma were analyzed using immunohistochemistry (IHC). IHC was used to detect ligand and receptor expression in the human ovarian cancer cells from Hey A8 and SKOV3ip1 growing in culture. Cells from these lines were also implanted orthotopically into the peritoneal cavity of nude mice. IHC was used to determine ligand and receptor expression in tumors that formed in the peritoneal cavity. Results. All 10 evaluable samples expressed both PDGF AA and BB on tumor cells. Tumor cells were positive for PDGF-Rα in 10/10 samples, PDGF-Rβ in 8/10 samples, p-PDGF-Rα in 6/10 samples, and p-PDGF-Rβ in 4/10 samples. p-PDGF-Rα was positive in 4/10 tumor-associated endothelial cell samples and p-PDGF-Rβ was positive in 3/10 samples. Human ovarian cancer cells expressed PDGF, PDGF-R, and p-PDGF-R when growing in culture or in the peritoneal cavity of nude mice. PDGF-R and p-PDGF-R were also present on tumor-associated endothelial cells as demonstrated by simultaneous staining with CD31 antibody. Conclusions. PDGF and the corresponding receptors were expressed in autochthonous human ovarian cancer lesions on both tumor cells and tumor-associated endothelial cells. The ligand and receptor were also present on Hey A8 and SKOV3ip1 human ovarian cancer cells growing in vitro and in the peritoneal cavity of nude mice.

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells.

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF alpha and beta receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.

The role of platelet derived growth factor in endomyocardial biopsies shortly after heart transplantation in relation to postoperative course.

Platelet-derived growth factor (PDGF) plays an important role in structural alterations of blood vessels after heart transplantation (HTx). The aim of this study was to clarify the effect of peritransplant injury and postoperative complications on the expression of PDGF ligand and receptor. Right ventricular endomyocardial biopsies were collected from 46 patients before implantation, and then 1 and 2 weeks after HTx. According to the clinical course in the first postoperative year and to histopathological evaluation (based on the standardised 'International Society for Heart and Lung Transplantation' grading system) three groups were formed: (a) clinical uneventful course; (b) histologically and/or serologically proven cardiac or systemic infections; and (c) acute rejection episodes > or =grade 3A. Both, infections and rejections were detectable after the second postoperative week. The expression of PDGF AA/BB and PDGF receptors alpha/beta was examined immunohistochemically. The infiltrating cells were characterised by using monoclonal antibodies against CD3, CD4, CD8, CD57 and CD68. Only endothelial cells revealed a relevant expression of PDGF ligand and receptor. Prior to implantation there was no or only weak reactivity of single cells for all PDGF factors. One week after HTx a significantly increased immunoreactivity of all PDGF factors was observed in all groups. Two weeks after HTx the expression of PDGF AA in the infection group and the expression of all four PDGF factors in the rejection group remained significantly elevated. In contrast, in the group with an uneventful course there was no statistical difference in the expression of all the four PDGF factors. Compared with the uneventful group, there were significantly more CD3+ cells in the infection and rejection group at all three time points. Two weeks after HTx, the rejection group showed a significantly elevated number of CD3+ cells compared to the values before implantation. Two weeks after HTx there were significant more CD68+ cells in the infection and rejection group compared with before implantation. One week after HTx the peritransplant injury predominantly influences the endothelial expression of PDGF ligand and receptor. In the first postoperative week, expression of PDGF could be detected. The persistence of evaluated PDGF expression might be of prognostic value in terms of a risk for either infection or rejection. These patients should be carefully monitored.

Role of p22phox in angiotensin II and platelet-derived growth factor AA induced activator protein 1 activation in vascular smooth muscle cells.

Reactive oxygen species (ROS) are involved in the transcriptional response to angiotensin (ANG) II. In this setting the role of NAD(P)H oxidase, an important source of ROS as second messengers, is not completely understood. In particular in human cells detailed insights into this mechanism are lacking. We investigated the role of ANG II and platelet-derived growth factor (PDGF) AA induced ROS generation derived from p22phox-containing NAD(P)H oxidase in the activation of activator protein (AP) 1 in human vascular smooth muscle cells (SMCs). Both ANG II and PDGF AA induced ROS generation in SMCs which was angiotensin type 1 receptor and PDGF alpha receptor dependent. Specific inhibition of the p22phox subunit of the NAD(P)H oxidase using either p22phox neutralizing antibody or p22phox antisense oligodeoxynucleotides (ODNs) attenuated both ANG II and PDGF AA induced ROS generation. Furthermore, PDGF AA but not ANG II induced p22phox mRNA expression. ANG II and PDGF AA both activated the redox-sensitive transcription factor AP-1, which was inhibited by p22phox antisense ODNs. These findings demonstrate that AP-1 activation in human SMCs in response to ANG II and PDGF AA is mediated via generation of p22phox-dependent ROS. This highlights the crucial role of the p22phox-containing NAD(P)H oxidase in the ANG II and PDGF AA induced signal transduction pathway.

Platelet-derived Growth Factor (PDGF) Receptor-α-activated c-Jun NH2-terminal Kinase-1 Is Critical for PDGF-induced p21WAF1/CIP1 Promoter Activity Independent of p53

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. PDGF AA functions as a "competent factor" that stimulates cell cycle entry but requires additional (progression) factors in serum to transit the cell cycle beyond the G1/S checkpoint. Unlike PDGF AA, PDGF B-chain (c-sis) homodimer (PDGF BB) and its viral counterpart v-sis can serve as both competent and progression factors. PDGF BB activates alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and induces phenotypic transformation in NIH 3T3 cells, whereas PDGF AA activates alpha-PDGFR only and fails to induce transformation. We showed previously that alpha-PDGFR antagonizes beta-PDGFR-mediated transformation through activation of stress-activated protein kinase-1/c-Jun NH2-terminal kinase-1, whereas both alpha-PDGFR and beta-PDGFR induce mitogenic signals. These studies revealed a striking feature of PDGF signaling; the specificity and the strength of the PDGF growth signal is modulated by alpha-PDGFR-mediated simultaneous activation of growth stimulatory and inhibitory signals, whereas beta-PDGFR mainly induces a growth-promoting signal. Here we demonstrate that PDGF BB activation of beta-PDGFR alone results in more efficient cell cycle transition from G1 to S phase than PDGF BB activation of both alpha-PDGFR and beta-PDGFR. PDGF AA activation of alpha-PDGFR or PDGF BB activation of both alpha- and beta-PDGFRs up-regulates expression of p21WAF1/CIP1, an inhibitor of cell cycle-dependent kinases and a downstream mediator of the tumor suppressor gene product p53. However, beta-PDGFR activation alone fails to induce p21WAF1/CIP1 expression. We also demonstrate that alpha-PDGFR-activated JNK-1 is a critical signaling component for PDGF induction of p21WAF1/CIP1 promoter activity. The ability of PDGF/JNK-1 to induce p21WAF1/CIP1 promoter activity is independent of p53, although the overall p21WAF1/CIP1 promoter activities are greatly reduced in the absence of p53. These results provide a molecular basis for differential regulation of the cell cycle and transformation by alpha- and beta-PDGFRs.

Role for TGF-beta1, FGF-2 and PDGF-AA in a myelination of CNS aggregate cultures enriched with macrophages.

The increase in myelin basic protein (MBP) synthesis observed in brain aggregate cultures supplemented with macrophages is reflected in elevated supernatant protein levels of the key promoters of oligodendrocyte proliferation, fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA), during the premyelinating phase. Although supernatant levels of transforming growth factor-beta1 (TGF-beta1), the most abundant growth factor produced at the transcriptional and translational levels by phagocytic macrophages, were reduced at this stage, it was the only growth factor for which mRNA expression was increased significantly in macrophage-enriched cultures. TGF-beta1, which supports oligodendrocyte differentiation, was increased in the supernatant of macrophage-enriched cultures only after the onset of myelinogenesis. Hence, standard cultures treated with TGF-beta1 during the premyelinating period reproduced effects of macrophage supplementation, inducing an increase in MBP synthesis and in PDGF-AA and FGF-2 bioavailability. A similar increase in MBP synthesis in PDGF-AA treated cultures emphasises its central role in oligodendrocyte progenitor proliferation. In contrast, FGF-2 blocked MBP synthesis in the cultures. In cultures treated with anti-TGF-beta1 antibody before or after the first detection of MBP, supernatant levels of TGF-beta1, FGF-2, and PDGF-AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF-beta1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF-beta1 in oligodendrocyte development and, as a source of TGF-beta1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.

Expression of platelet-derived growth factor-alpha alpha receptor is associated with tumor progression in clear cell renal cell carcinoma.

Platelet-derived growth factors (PDGFs) exert their biologic function by binding to 3 different tyrosine kinase receptor isoforms. Especially the PDGF-alpha alpha receptor binds PDGF proteins with high specificity, which results in growth stimulation. The expression of the receptor and its ligand was studied in human renal cell carcinomas (RCCs) by immunohistochemical analysis, and the expression of PDGF-alpha alpha receptor and PDGF-AA was correlated with clinicopathologic parameters of patients with clear cell RCC (CCRCC). In CCRCC, the mean expression of PDGF-alpha alpha receptor and PDGF-AA was 38.8% (range, 0.0%-96.0%) and 18.4% (range, 0.0%-90.0%), respectively. PDGF-alpha alpha receptor expression was significantly higher in grade 3 and grade 4 tumors compared with grade 1 and grade 2 tumors (P = .027; Mann-Whitney test), and high receptor expression correlated with tumor progression in univariate analysis (P = .0253; log-rank test), while PDGF-AA expression had no prognostic influence on the outcome of patients with CCRCC. Therefore, immunohistochemical detection of high PDGF-alpha alpha receptor expression in CCRCC is associated with adverse outcomes. Furthermore, the PDGF receptor-factor interaction loop may be considered as a possible target for novel therapeutic strategies.