06-127 Sigma-AldrichAnti-PDGF Antibody, neutralizing

There is increasing evidence that proteins in tubular fluid are "nephrotoxic." In vivo it is difficult to study protein loading of tubular epithelial cells in isolation, i.e., without concomitant glomerular damage or changes of renal hemodynamics, etc. Recently, a unique amphibian model has been described which takes advantage of the special anatomy of the amphibian kidney in which a subset of nephrons drains the peritoneal cavity (open nephrons) so that intraperitoneal injection of protein selectively causes protein storage in and peritubular fibrosis around open but not around closed tubules. There is an ongoing debate as to what degree albumin per se is nephrotoxic and whether modification of albumin alters its nephrotoxicity. We tested the hypothesis that carbamylation and glycation render albumin more nephrotoxic compared with native albumin and alternative albumin modifications, e.g., lipid oxidation and lipid depletion. Preparations of native and modified albumin were injected into the axolotl peritoneum. The kidneys were retrieved after 10 days and studied by light microscopy as well as by immunohistochemistry [transforming growth factor (TGF)-β, PDGF, NF-κB, collagen I and IV, RAGE], nonradioactive in situ hybridization, and Western blotting. Two investigators unaware of the animal groups evaluated and scored renal histology. Compared with unmodified albumin, glycated and carbamylated albumin caused more pronounced protein storage. After no more than 10 days, selective peritubular fibrosis was seen around nephrons draining the peritoneal cavity (open nephrons), but not around closed nephrons. Additionally, more intense expression of RAGE, NF-κB, as well as PDGF, TGF-β, EGF, ET-1, and others was noted by histochemistry and confirmed by RT-PCR for fibronectin and TGF-β as well as nonradioactive in situ hybridization for TGF-β and fibronectin. The data indicate that carbamylation and glycation increase the capacity of albumin to cause tubular cell damage and peritubular fibrosis.

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions of neurodegenerative diseases; however, the precise mechanisms by which MSCs migrate remain to be elucidated. In this study, we carried out an in vitro migration assay to investigate the chemoattractive factors for MSCs in the brains of prion-infected mice. The migration of immortalized human MSCs (hMSCs) was reduced by their pretreatment with antibodies against the chemokine receptors, CCR3, CCR5, CXCR3, and CXCR4 and by pretreatment of brain extracts of prion-infected mice with antibodies against the corresponding ligands, suggesting the involvement of these receptors, and their ligands in the migration of hMSCs. In agreement with the results of an in vitro migration assay, hMSCs in the corpus callosum, which are considered to be migrating from the transplanted area toward brain lesions of prion-infected mice, expressed CCR3, CCR5, CXCR3, and CXCR4. The combined in vitro and in vivo analyses suggest that CCR3, CCR5, CXCR3, and CXCR4, and their corresponding ligands are involved in the migration of hMSCs to the brain lesions caused by prion propagation. In addition, hMSCs that had migrated to the right hippocampus of prion-infected mice expressed CCR1, CX3CR1, and CXCR4, implying the involvement of these chemokine receptors in hMSC functions after chemotactic migration. Further elucidation of the mechanisms that underlie the migration of MSCs may provide useful information regarding application of MSCs to the treatment of prion diseases.

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.

The development of normal breast tissue and the pathogenesis of various tumors are influenced by growth factor-mediated epithelial-stromal interactions. Similar interactions may occur in fibroepithelial breast tumors. We have studied the expression of platelet-derived growth factor (PDGF) and PDGF beta receptor (PDGFRbeta) in 46 phyllodes tumors (18 benign, 15 borderline, 13 malignant), 11 fibroadenomas, and 6 samples of normal breast. There was neoplastic stromal cell positivity for PDGFRbeta in almost 50% of phyllodes tumors and for PDGF in 24%. Both were associated with prominent nuclear pleomorphism (P<.01), PDGF with high grade (P<.01), and a higher mean Ki-67 labeling index (P = .013), and PDGFRbeta with conspicuous stromal overgrowth (P<.01). Co-positivity for stromal PDGF and PDGFRbeta was found in 15% of phyllodes tumors, and for epithelial PDGF and stromal PDGFRbeta in 43%. Both types of co-positivity were associated with prominent nuclear pleomorphism and the latter type with conspicuous stromal overgrowth (all P<.01). Follow-up of 41 phyllodes tumors showed that disease-related death was associated with established histologic features of malignancy including mitotic count, stromal overgrowth, an infiltrative tumor margin, and nuclear pleomorphism. In addition, stromal PDGFRbeta positivity (P =.013) and epithelial PDGF/stromal PDGFRbeta co-positivity (P =.0075) were associated with disease-related death. Stromal PDGF and PDGFRbeta expression in fibroadenomas was less common and less extensive (P<.05) than in phyllodes tumors. The results suggest that PDGF influences the pathogenesis of fibroepithelial breast tumors and that PDGF-dependent paracrine and autocrine mechanisms may operate. Also, it is possible that assessment of PDGF and PDGFRbeta expression could contribute to the management of these tumors in the future.

When platelet-derived growth factor (PDGF) binds to its receptor on a quiescent fibroblast or smooth muscle cell, it stimulates a remarkably diverse group of biochemical responses, including changes in ion fluxes, activation of several kinases, alterations in cell shape, increased transcription of a number of genes, and stimulation of enzymes that regulate phospholipid metabolism. These and other reactions culminate, hours later, in DNA replication and cell division. How does the receptor for PDGF recognize and bind its specific ligand and then transduce this signal across the cell membrane via a single membrane-spanning region? Which of the immediate cellular responses are directly involved in the biochemical pathways that lead to DNA synthesis? How does the PDGF receptor trigger a diverse group of responses? Recent studies of the PDGF receptor have provided insight into these issues.

An increasing number of polypeptide growth factors have been identified that regulate not only cell proliferation but an extraordinary range of cell activities, including matrix protein deposition and resolution, the maintenance of cell viability, cell differentiation, inflammation, and tissue repair. Normal cells appear to require growth factors for proliferation and for maintenance of viability. Cells that secrete a polypeptide growth factor have an advantage in growth. These factors can act either externally through cell surface receptors or perhaps internally during the transport of receptors and growth factors through the ER and Golgi, causing autocrine stimulation of cell growth. Depending on the cell type, growth factors can also be potent inhibitors of cell growth rather than stimulating growth, and the effects can depend on the presence or absence of other growth factors. Platelet-derived growth factor has been shown to be nearly identical to the product of the v-sis gene of the simian sarcoma virus, which appears to cause cell transformation through its interactions with the PDGF receptor activating the tyrosine kinase activity of the PDGF receptor. Similarly, two proto-oncogenes, c-erbB and c-fms, encode growth factor receptors. The EGF receptor activity of the v-erb oncogene product appears to be constitutively activated without the need for growth factor, perhaps because of the truncation at the amino terminus deleting the EGF binding domain. The induction of the myc and the fos proteins by growth factor stimulation of quiescent cells, as well as the potential for the p21 product of the ras oncogene to act as an intermediate in transducing adrenergic signals, provide direct evidence that these pathways are important for stimulation of cell growth. Cells transformed by the v-sis oncogene always appear to bear PDGF cell surface receptors, which suggests that this oncogene has a specific requirement of the PDGF receptor for transformation. In contrast, cells transformed by the v-erbB and v-fms oncogenes are not stimulated by EGF or by CSF-1. Thus it seems likely that the tyrosine kinase activity of the corresponding receptor is ubiquitously expressed in these cases. Major questions remain unanswered. In particular, what are the mechanisms by which growth factors initiate pathways leading to DNA synthesis? What are the physiological substrates of the growth factor receptor tyrosine kinase? Considerable effort also is needed to further define the cellular specificity of the different growth factors, particularly within intact tissues, and to determine how the various growth factors interact.(ABSTRACT TRUNCATED AT 400 WORDS)