BACKGROUND

In various heart disease paradigms, atria show stronger fibrotic responses than ventricles. The possibility that atrial and ventricular fibroblasts respond differentially to pathological stimuli has not been examined.

RESULTS

We compared various morphological, secretory, and proliferative response indexes of canine atrial versus ventricular fibroblasts. Cultured atrial fibroblasts showed faster cell surface area increases, distinct morphology at confluence, and greater alpha-smooth muscle actin expression than ventricular fibroblasts. Atrial fibroblast proliferation ([(3)H]thymidine incorporation) responses were consistently greater for a range of growth factors, including fetal bovine serum, platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, endothelin-1, and transforming growth factor-beta(1). Normal atrial tissue showed larger myofibroblast density compared with ventricular tissue, and the difference was exaggerated by congestive heart failure. Congestive heart failure atria showed larger fractions of fibroblasts in mitotic phases compared with ventricles and displayed enhanced gene expression of fibroblast-selective markers (collagen-1, collagen-3, fibronectin-1). Gene microarrays revealed 225 differentially expressed transcript probe sets between paired atrial and ventricular fibroblast samples, including extracellular matrix (eg, fibronectin, laminin, fibulin), cell signaling (PDGF, PDGF receptor, angiopoietin, vascular endothelial growth factor), structure (keratin), and metabolism (xanthine dehydrogenase) genes, identifying PDGF as a candidate contributor to atrial-ventricular fibroblast differences. PDGF receptor gene expression was greater in normal atrium compared with ventricle, and congestive heart failure differentially enhanced atrial versus ventricular PDGF and PDGF receptor gene expression. PDGF receptor protein expression and alpha-smooth muscle actin protein expression were enhanced in isolated congestive heart failure fibroblasts. The PDGF receptor tyrosine kinase inhibitor AG1295 eliminated fetal bovine serum- and transforming growth factor-beta(1)-stimulated atrial-ventricular fibroblast proliferative response differences.

CONCLUSIONS

Atrial fibroblasts behave differently than ventricular fibroblasts over a range of in vitro and in vivo paradigms, with atrial fibroblasts showing enhanced reactivity that may explain greater atrial fibrotic responses. PDGF signaling is particularly important for atrium-selective fibroblast responses and may represent a novel target for arrhythmogenic atrial structural remodeling prevention.

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.

Tumor stroma formation results from the interaction of tumor cells and their products with the host and certain of its normal defense mechanisms, particularly the clotting and fibrinolytic systems. It is a process in which tumor cells render local venules and veins hyperpermeable with the result that fibrinogen and other proteins extravasate and clot, forming an extravascular crosslinked fibrin gel. Coagulation is mediated by an interaction between extravasated plasma clotting factors and tumor-associated and perhaps other tissue procoagulants. Parallel activation of the fibrinolytic system leads to substantial fibrin turnover, but fibrin nonetheless accumulates in amounts, variable from tumor to tumor, that are sufficient to provide a provisional stroma. This provisional stroma imposes on tumor cells a structure that persists even as tumor cells multiply and as the fibrin provisional stroma is replaced by mature connective tissue. The provisional fibrin stroma also serves to regulate the influx of macrophages, and perhaps other inflammatory cells, but at the same time, and in ways that are not fully understood, facilitates the inward migration of new blood vessels and fibroblasts, integral components of mature tumor stroma. Ascites tumors differ from solid tumors in that fibrin gel is not ordinarily deposited in body cavities and, as a result, there is no provisional stroma to impose an initial structure. Tumor stroma generation resembles the process of wound healing in many respects. However, it differs in the mechanism of its initiation, and in the apparent lack of a role for platelets. It also differs fundamentally in that invading tumor cells continually render new vessels hyperpermeable to plasma, thus perpetuating the cycle of extravascular fibrin deposition. In this sense, tumors behave as wounds that do not heal. Largely neglected in this review has been discussion of the numerous cytokines, mitogens, and growth factors that are widely believed to play important roles in tumor angiogenesis and wound healing; i.e., PDGF, FGF, EGF, TGF alpha, TGF beta, TNF, interferons, etc. This omission has been intentional, and for two reasons. First, these cytokines have already received considerable attention [100,123-128]. Second, it is not yet clear how closely the actions of these molecules, as described in vitro, relate to their functions in vivo. At present we are deluged with a surfeit of factors that have the capacity to induce new blood vessel formation in angiogenesis assays; these factors include not only peptides but lipids and even ions [126,129-131].(ABSTRACT TRUNCATED AT 400 WORDS)

The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.

Calcifications in the basal ganglia are a common incidental finding and are sometimes inherited as an autosomal dominant trait (idiopathic basal ganglia calcification (IBGC)). Recently, mutations in the PDGFRB gene coding for the platelet-derived growth factor receptor β (PDGF-Rβ) were linked to IBGC. Here we identify six families of different ancestry with nonsense and missense mutations in the gene encoding PDGF-B, the main ligand for PDGF-Rβ. We also show that mice carrying hypomorphic Pdgfb alleles develop brain calcifications that show age-related expansion. The occurrence of these calcium depositions depends on the loss of endothelial PDGF-B and correlates with the degree of pericyte and blood-brain barrier deficiency. Thus, our data present a clear link between Pdgfb mutations and brain calcifications in mice, as well as between PDGFB mutations and IBGC in humans.

Activation of the platelet-derived growth factor (PDGF) beta-receptor results in motility responses in the forms of membrane ruffling and chemotaxis. Porcine aortic endothelial cells expressing the PDGF beta-receptor or a chimeric fibroblast growth factor (FGF) receptor, in which the endogenous kinase insert was replaced with the corresponding region from the PDGF beta-receptor, migrated efficiently towards a concentration gradient of PDGF-BB and bFGF, respectively, and exhibited both pronounced edge ruffling and circular membrane ruffling in response to ligand-stimulation. The wildtype FGF receptor-1 showed weak or no response in these assays. Further analyses were conducted on mutant receptors, in which tyrosine residues that can serve as autophosphorylation sites and thereby mediate interactions with specific signal transduction molecules, were changed to phenylalanine residues. Each one of the analysed mutants were mitogenically active, however, a mutant in which Tyr740 and Tyr751 were replaced failed to mediate ruffling and chemotaxis. These two residues are implicated in the binding of phosphatidylinositol 3' kinase. The notion that this enzyme is involved in PDGF beta-receptor-induced cell motility is furthermore supported by the finding that another mutant, in which Met743 and Met754 were replaced, and which failed to interact with phosphatidylinositol 3' kinase, was also unable to mediate motility responses.

A consistent response to liver injury is the activation of resident mesenchymal cells known as lipocytes (Ito, fat-storing cells) into a proliferating cell type. In cultured lipocytes, platelet-derived growth factor (PDGF) is the most potent proliferative cytokine, but requires the activation-dependent expression of its receptor protein (Friedman, S. L., and M. J. P. Arthur. 1989. J. Clin. Invest. 84:1780-1785); the role of PDGF receptor (PDGFR) in liver injury is unknown. We have examined PDGFR gene expression in freshly isolated lipocytes during liver injury and correlated these findings with a culture model of cellular activation. Whereas lipocytes from normal rats had no detectable transcript for the beta-PDGFR subunit, this mRNA was induced within 1 h after a dose of carbon tetrachloride (CCl4). In contrast, alpha subunit mRNA was detected in normal cells, but was unchanged after liver injury. Similar results were observed in lipocytes from bile duct-obstructed rats, although beta-PDGFR induction was less marked. By immunoblot, induction of beta-PDGFR protein in lipocytes isolated from CCl4-treated animals correlated with mRNA increases. In contrast to lipocytes, endothelial cells from normal liver expressed low levels of alpha- and beta-receptor subunit mRNA, which did not increase with injury. Using a beta-PDGFR antibody, receptor protein could be identified within fibrotic septa in CCl4-treated animals in regions where cells expressed proliferating cell nuclear antigen (PCNA). In cultured lipocytes activated by growth on uncoated plastic, beta-PDGFR transcripts appeared within 3 d after plating, which coincided with the onset of cellular proliferation. In contrast, quiescent cells in suspension culture had no detectable beta-PDGFR mRNA. These results indicate that beta-PDGF receptor induction by lipocytes is an early event during hepatic injury in vivo and in primary culture.

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.

The origin of myofibroblasts and the factors promoting their differentiation during liver fibrogenesis remain uncertain. During biliary-type fibrogenesis, the proliferation and chemoattraction of hepatic stellate cells (HSC) toward bile ducts is mediated by platelet-derived growth factor (PDGF), while myofibroblastic conversion of peribiliary cells distinct from HSC also occurs. We herein examined the phenotype of these peribiliary myofibroblasts as compared with myofibroblastic HSC and tested whether their differentiation was affected by PDGF. Biliary-type liver fibrogenesis was induced by common bile duct ligation in rats. After 48 hours, periductular fibrosis in portal tracts colocalized with smooth muscle alpha-actin-immunoreactive myofibroblasts, the majority of which were desmin negative. Simultaneously, in sinusoids, desmin immunoreactivity was induced in a large number of HSC, which were smooth muscle alpha-actin negative. Cultures of peribiliary myofibroblasts were expanded from isolated bile duct segments and compared with myofibroblastic HSC. Peribiliary myofibroblasts outgrowing from bile duct segments expressed smooth muscle alpha-actin, alpha1 (I) collagen mRNA, and PDGF receptor-beta subunit. Desmin immunoreactivity gradually decreased in cultured peribiliary myofibroblasts, contrasting with constant labeling of all myofibroblastic HSC. In addition, IL-6 expression in peribiliary myofibroblasts was up to 100-fold lower than in myofibroblastic HSC, whereas the expression of the complement-activating protease P100 in both cell types showed little difference and that of the extracellular matrix component fibulin 2 was similar. The expression of smooth muscle alpha-actin protein in cultured peribiliary myofibroblasts was stimulated by PDGF-BB and inhibited by STI571, a PDGF receptor tyrosine kinase inhibitor, whereas in bile duct-ligated rats, the administration of STI571 caused a significant decrease in peribiliary smooth muscle alpha-actin immunoreactivity, and to a lesser extent, a decrease in peribiliary fibrosis. These results indicate that peribiliary cells distinct from HSC undergo a PDGF-mediated conversion into myofibroblasts expressing IL-6 at lower levels than myofibroblastic HSC and contribute to the initial formation of biliary-type liver fibrosis.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.

Fractured bones heal by a cascade of cellular events in which mesenchymal cells respond to unknown regulators by proliferating, differentiating, and synthesizing extracellular matrix. Current concepts suggest that growth factors may regulate different steps in this cascade (10). Recent studies suggest regulatory roles for PDGF, aFGF, bFGF, and TGF-beta in the initiation and the development of the fracture callus. Fracture healing begins immediately following injury, when growth factors, including TGF-beta 1 and PDGF, are released into the fracture hematoma by platelets and inflammatory cells. TGF-beta 1 and FGF are synthesized by osteoblasts and chondrocytes throughout the healing process. TGF-beta 1 and PDGF appear to have an influence on the initiation of fracture repair and the formation of cartilage and intramembranous bone in the initiation of callus formation. Acidic FGF is synthesized by chondrocytes, chondrocyte precursors, and macrophages. It appears to stimulate the proliferation of immature chondrocytes or precursors, and indirectly regulates chondrocyte maturation and the expression of the cartilage matrix. Presumably, growth factors in the callus at later times regulate additional steps in repair of the bone after fracture. These studies suggest that growth factors are central regulators of cellular proliferation, differentiation, and extracellular matrix synthesis during fracture repair. Abnormal growth factor expression has been implicated as causing impaired or abnormal healing in other tissues, suggesting that altered growth factor expression also may be responsible for abnormal or delayed fracture repair. As a complete understanding of fracture-healing regulation evolves, we expect new insights into the etiology of abnormal or delayed fracture healing, and possibly new therapies for these difficult clinical problems.

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately 5.7 kb) and a minor (approximately 4.8 kb) transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an approximately equal to 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF (i.e., PDGF dimers composed of two B chains or an A and a B chain). Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.

Brainstem gliomas (BSG) are a rare group of central nervous system tumors that arise mostly in children and usually portend a particularly poor prognosis. We report the development of a genetically engineered mouse model of BSG using the RCAS/tv-a system and its implementation in preclinical trials. Using immunohistochemistry, we found that platelet-derived growth factor (PDGF) receptor alpha is overexpressed in 67% of pediatric BSGs. Based on this observation, we induced low-grade BSGs by overexpressing PDGF-B in the posterior fossa of neonatal nestin tv-a mice. To generate high-grade BSGs, we overexpressed PDGF-B in combination with Ink4a-ARF loss, given that this locus is commonly lost in high-grade pediatric BSGs. We show that the likely cells of origin for these mouse BSGs exist on the floor of the fourth ventricle and cerebral aqueduct. Irradiation of these high-grade BSGs shows that although single doses of 2, 6, and 10 Gy significantly increased the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei, only 6 and 10 Gy significantly induce cell cycle arrest. Perifosine, an inhibitor of AKT signaling, significantly induced TUNEL-positive nuclei in this high-grade BSG model, but in combination with 10 Gy, it did not significantly increase the percent of TUNEL-positive nuclei relative to 10 Gy alone at 6, 24, and 72 hours. Survival analysis showed that a single dose of 10 Gy significantly prolonged survival by 27% (P = 0.0002) but perifosine did not (P = 0.92). Perifosine + 10 Gy did not result in a significantly increased survival relative to 10 Gy alone (P = 0.23). This PDGF-induced BSG model can serve as a preclinical tool for the testing of novel agents.

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.

Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/β-catenin signaling responses that are markedly up-regulated following kidney injury. Dickkopf-related protein 1 (DKK-1), a ligand for the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP-5 and LRP-6) and an inhibitor of WNT/β-catenin signaling, effectively inhibits pericyte activation, detachment, and transition to myofibroblasts in vivo in response to kidney injury, resulting in attenuated fibrogenesis, capillary rarefaction, and inflammation. DKK-1 blocks activation and proliferation of established myofibroblasts in vitro and blocks pericyte proliferation to PDGF, pericyte migration, gene activation, and cytoskeletal reorganization to TGF-β or connective tissue growth factor. These effects are largely independent of inhibition of downstream β-catenin signaling. DKK-1 acts predominantly by inhibiting PDGF-, TGF-β-, and connective tissue growth factor-activated MAPK and JNK signaling cascades, acting via LRP-6 with associated WNT ligand. Biochemically, LRP-6 interacts closely with PDGF receptor β and TGF-β receptor 1 at the cell membrane, suggesting that it may have roles in pathways other than WNT/β-catenin. In summary, DKK-1 blocks many of the changes in pericytes required for myofibroblast transition and attenuates established myofibroblast proliferation/activation by mechanisms dependent on LRP-6 and WNT ligands but not the downstream β-catenin pathway.

Previously we showed that interleukin 1 beta stimulates the conversion of sphingomyelin to ceramide in the caveolae fraction of normal human fibroblasts. The ceramide, in turn, blocked platelet-derived growth factor (PDGF) stimulated DNA synthesis. We now present evidence that the PDGF receptor initiates signal transduction from caveolae. Cell fractionation and immunocytochemistry show caveolae to be the principal location of PDGF receptors at the cell surface. Multiple caveolae proteins acquire phosphotyrosine when PDGF binds to its receptor, but the hormone appears to have little effect on the tyrosine phosphorylation of non-caveolae membrane proteins. Five proteins known to interact with the phosphorylated receptor were found to be highly enriched in caveolae membrane. PDGF caused the concentration of three of these proteins to significantly increase in the caveolae fraction. Finally, PDGF stimulated the association of a 190-kDa phosphoprotein with the caveolae marker protein, caveolin. Therefore, ceramide may modulate PDGF receptor function directly in caveolae.

Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to cell surface heparan sulfate proteoglycan (HSPG). This was demonstrated in (a) the binding of 125I-HB-EGF to mutant CHO cells deficient in HS production was diminished by 70% compared to wild-type CHO cells, (b) the binding of 125I-HB-EGF to CHO cells and bovine aortic smooth muscle cells (BASMC) was diminished 80% by heparitinase or chlorate treatment, and (c) 125I-EGF did not bind to CHO cells and its binding to BASMC was not diminished at all by heparitinase and only slightly by chlorate treatment. Accordingly, the role of HB-EGF interactions with HSPG in modulating bioactivity was examined. Heparitinase or chlorate treatment of BASMC diminished the ability of HB-EGF to stimulate BASMC migration by 60-80%. A similar inhibition of migration occurred when BASMC were treated with a synthetic peptide (P21) corresponding to the sequence of the putative heparin-binding domain of HB-EGF. As a control for BASMC viability, and for specificity, it was found that heparitinase and P21 did not inhibit at all and chlorate inhibited only slightly the stimulation of BASMC migration by PDGF AB. Since heparitinase, chlorate, and P21 treatment also diminished by 70-80% the cross-linking of 125I-HB-EGF to the EGF receptor, it was concluded that the interaction of HB-EGF, via its heparin-binding domain, with cell surface HSPG was essential for its optimal binding to the EGF receptor on BASMC and hence for its optimal ability to stimulate migration.

Vascular endothelial cells have a central role in various pathophysiological responses such as acute inflammation, wound healing and atherogenesis. The anatomical position of endothelial cells between blood leukocytes and the surrounding vascular smooth muscle cells or stromal fibroblasts may intensify and focus the effects of released endothelial cell products. Endothelial cells in culture produce a platelet-derived growth factor (PDGF)-like mitogen. PDGF purified from platelets is a basic protein with an apparent relative molecular mass (Mr) of approximately 30,000 (reviewed in refs 2, 3) and is believed to comprise two polypeptide chains, PDGF-A and PDGF-B (also referred to as PDGF-1 and PDGF-2; refs 5, 6). Sequence analysis of PDGF B chain has revealed a striking homology with the predicted sequence of p28sis, the transforming protein of simian sarcoma virus. sis-Homologous transcripts have been detected by Northern blot analysis of RNA from cultured endothelial cells. However, there are no structural data available on either the protein product or the messenger RNA to establish the identity of the endothelial-derived mitogen with either chain of PDGF. Here we report the isolation and complete sequence analysis of a sis-homologous complementary DNA clone from human endothelial cells, providing an opportunity to study the structure of sis as transcribed by a normal (untransformed) cell. Our results establish that normal human endothelial cells in culture express the B chain of PDGF, and that endothelial-derived PDGF B chain is synthesized as a predicted precursor polypeptide of Mr 27,281.

Signals transmitted from mesenchyme to epithelia or vice versa constitute the basis of reciprocal epithelial-mesenchymal interactions. As a first step toward understanding epithelial-mesenchymal interactions on the ocular surface where the transit amplifying cell-containing corneal epithelium is anatomically separated from the stem cell-containing limbal epithelium, we sought to characterize the expression patterns of cytokines and their receptors by primary epithelial and early-passaged fibroblast cultures of human cornea and limbus. Northern hybridization with oligonucleotide and cDNA probes to a total of 25 cytokines and 12 of their receptors revealed that the positively expressed cytokines could be divided into the following four patterns. Type I: TGF-alpha, IL-1 beta, and PDGF-B were expressed exclusively by epithelial cells but their respective receptors EGFR and IL-1R were predominantly and PDGFR-beta was exclusively expressed by fibroblasts. Type II: IGF-I, TGF-beta 1, -beta 2, LIF, and bFGF, and their receptors were expressed by both epithelial cells and fibroblasts. FGFR-1 (flg) and FGFR-2 (bek) were expressed more by fibroblasts and bFGF was expressed more by corneal than limbal epithelial cells. Type III: keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were expressed exclusively by fibroblasts and their respective receptors, KGFR and c-met, were predominantly expressed by epithelial cells. Combined with RT-PCR, the quantity of KGF and KGFR transcripts was highest in limbal fibroblasts and epithelial cells, respectively. In contrast, the quantity of HGF and HGFR (c-met) transcripts was highest in corneal fibroblasts and epithelial cells, respectively. Type IV: M-CSF and IL-8 were expressed by fibroblasts and/or epithelial cells but their receptors were not expressed by epithelial cells nor fibroblasts, but by immune or inflammatory cells. In addition to these potential paracrine actions, autocrine actions mediated by TGF-alpha/EGFR, IL-1 beta/IL1-R, and bFGF/FGFR-1 were more expressed by corneal than limbal epithelial cells. Immunofluorescence staining on human corneoscleral cryosections confirmed that EGFR and bFGF were not expressed by the limbal basal epithelium, but expressed strongly by the corneal epithelium, a pattern consistent with Northern hybridization. These results indicate that ocular surface epithelial cells and fibroblasts can express a myriad of cytokines, among which the first three patterns constitute the network of potential epithelial-mesenchymal cytokine dialogues. The difference of certain cytokine expression between corneal and limbal regions suggests that this network participates in normal epithelial growth and differentiation, and plays an important role in wound healing.

Hypoxic states are associated with abnormal proliferation and constriction of the smooth muscle cells surrounding the distal vessels of the lung. In hypoxic as well as in normal states, the endothelial cell layer may play a key role in controlling smooth muscle tone by secreting a number of vasoactive agents. Platelet-derived growth factor (PDGF), produced by endothelial cells, is a major growth factor for vascular smooth muscle cells and a powerful vasoconstrictor. It consists of a disulfide-linked dimer of two related peptides, A and B, that are products of two different genes. We found that hypoxic conditions (0-3% oxygen environments) significantly increased PDGF-B mRNA in cultured human umbilical vein endothelial cells by enhancing the transcriptional rate of this gene. This increase was inversely proportional to oxygen tension and was reversible upon reexposure of cells to a 21% oxygen atmosphere. mRNA levels of PDGF-A were not affected nor was the overall rate of cellular gene transcription increased in response to hypoxia. These studies indicate that endothelial cells are not only capable of sensing oxygen tension, but are also able to discriminate and respond to even small differences in oxygen tension resulting in dramatic upregulation of the PDGF-B chain gene.

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.

Established cell lines derived from human malignant astrocytomas typically express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could form an autocrine loop. In this study, we screened for the essential components of a PDGF autocrine loop in fresh surgical isolates of human astrocytomas, using in situ hybridization and immunohistochemical techniques. Eight malignant astrocytomas (6 glioblastomas and 2 anaplastic astrocytomas), 5 low-grade astrocytomas and 4 non-neoplastic glial specimens (mesial temporal sclerosis) were evaluated. Malignant astrocytomas, and to a lesser extent low-grade astrocytomas, expressed more PDGF-A and PDGF-B than non-neoplastic glia. PDGF-alpha-receptor expression was elevated both in malignant and in low-grade astrocytomas. These data support the argument that PDGF autocrine loops contribute to the unregulated growth of human astrocytomas. Expression of PDGF and PDGF receptor in low-grade astrocytomas suggests that activation of PDGF autocrine loops may be an early event in the pathogenesis of malignant astrocytomas.

In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-beta is present locally shortly after wounding, but not unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor-beta 1 (TGF-beta 1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-beta resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in wound healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-alpha had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-beta release during the wound-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system.