Platelet-derived growth factor (PDGF) is encoded by separate genes for two possible subunit chains (A-chain and B-chain) which can form three possible dimers (AA, AB, and BB). We have recently presented evidence that multiple forms of PDGF receptor exist which distinguish between these isoforms (Hart, C. H., Forstrom, J. W., Kelley, J. D., Smith, R. A., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1988) Science 240, 1529-1531). We used this specificity to determine the amount of PDGF from different sources which is able to bind to each class of receptor and found that each source had a characteristic isoform composition. Levels of total PDGF activity in sera from different species ranged more than 15-fold, from less than 1 ng/ml in dog, chicken, pig, and calf, to greater than 13 ng/ml in mouse and human. Despite these differences in PDGF content, the total mitogenic activities of the sera were comparable indicating that the relative importance of PDGF as a serum mitogen may vary considerably between species. Analysis of the total PDGF into the amounts of each isoform revealed great differences in composition. PDGF-BB constitutes only about 15% of the total binding activity in human PDGF purified by the method of Raines and Ross (Raines, E. W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160) but is the predominant isoform in whole blood serum from all other species. In contrast to serum, medium conditioned by cultured PDGF-secreting cell types contained no detectable PDGF-BB except in two cases: medium conditioned by vascular endothelial cells and by cells transformed by simian sarcoma virus. The existence of isoform-specific PDGF receptors and the large variation in PDGF isoform composition dependent upon source may provide an important mechanism through which the effects of PDGF can be targeted to different cell types and/or toward eliciting different cell responses.

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 microM serotonin with increased incorporation of [3H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 microM. At a concentration of 1 microM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approximately 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta-receptors in human diploid foreskin AG 1518 fibroblasts. A transient tyrosine phosphorylation of PDGF beta-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not on poly-L-lysine. In contrast EGF or PDGF alpha-receptors were not phosphorylated on tyrosine residues under these conditions. Tyrosine phosphorylation of PDGF beta-receptors induced by plating on collagen type I was inhibited by cytochalasin D and herbimycin A, unaffected by cycloheximide and enhanced by orthovanadate. Furthermore, a transient phosphorylation of PDGF beta-receptors occurred when AG 518 fibroblasts were cultured in three-dimensional collagen lattices or exposed to external strain exerted through centrifugation. The latter effect was evident already after two minutes. Clustering of cell surface beta1 integrins led to PDGF beta-receptor phosphorylation both in suspended and firmly attached AG 1518 fibroblasts. Plating of cells on collagen type I, fibronectin, and anti-beta1-integrin IgG resulted in the formation of PDGF beta-receptor aggregates as detected by immunofluorescence. Suramin or anti-PDGF-BB IgG had no effect on the plating-induced tyrosine phosphorylation of PDGF beta-receptors. PDGF-B chain mRNA, or protein, were not detected in AG 1518 fibroblasts. Our data suggest that a ligand-independent PDGF beta-receptor activation during cell adhesion and early phases of cell spreading is involved in integrin-mediated signaling in fibroblasts, and constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction.

Protein kinase D (PKD) is a serine/threonine protein kinase that is activated by phorbol esters via protein kinase C in intact cells. To assess the physiological significance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bombesin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-transfected with PKD and bombesin/GRP receptor expression constructs. No inducible kinase activity was demonstrated when COS-7 cells were transfected with a kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the protein kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and in porcine aortic endothelial cells stably transfected with PDGF-beta receptors. Treatment with GF 1092030X or Ro 31-8220 inhibited PKD activation induced by PDGF. Thus, our results indicate that PKD is activated by multiple signaling peptides through a protein kinase C-dependent signal transduction pathway in a variety of cell types.

Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that PDGF (platelet-derived growth factor) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux and mitochondrial oxygen consumption were measured after treatment of primary rat aortic VSMCs (vascular smooth muscle cells) with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K (phosphoinositide 3-kinase) inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, LDH (lactate dehydrogenase) protein levels and activity were significantly increased after PDGF treatment. Moreover, substitution of L-lactate for D-glucose was sufficient to increase mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the LDH inhibitor oxamate. These results suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMCs in the diseased vasculature.

We have previously demonstrated the cardioprotective effects of exosomes derived from mesenchymal stem cells (MSCs). It is well known that the activation of Akt is involved in stem cell-induced cardioprotection. In the present study, we investigated whether exosomes released from Akt-overexpressing MSCs showed a beneficial effect on cardioprotection and angiogenesis. MSCs were collected from human umbilical cord (hucMSCs), and Akt was transfected into hucMSCs (Akt-hucMSCs) by using an adenovirus transfection system. Exosomes were isolated from control hucMSCs (Exo) and Akt-hucMSCs (Akt-Exo). An acute myocardial infarction model was created by ligation of the left anterior decedent coronary artery (LAD) in rats. Various source exosomes (400 µg of protein) were infused via the tail vein immediately after LAD ligation. The cardiac function was evaluated by using echocardiography after different treatments for 1 and 5 weeks, respectively. Endothelial cell proliferation, migration, and tube-like structure formation, as well as chick allantoic membrane assay, were used to evaluate the angiogenetic effects of Akt-Exo. The results indicated that cardiac function was significantly improved in the animals treated with Akt-Exo. In addition, Akt-Exo significantly accelerated endothelial cell proliferation and migration, tube-like structure formation in vitro, and blood vessel formation in vivo. The expression of platelet-derived growth factor D (PDGF-D) was significantly upregulated in Akt-Exo. However, the angiogenesis was abrogated in endothelial cells treated with the exosomes obtained from MSCs transfected with PDGF-D-siRNA. Our studies suggest that exosomes obtained from Akt-modified hucMSCs are more effective in myocardial infarction therapy through promoting angiogenesis. PDGF-D plays an important role in Akt-Exo-mediated angiogenesis. Stem Cells Translational Medicine 2017;6:51-59.

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet-derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)beta gene by Cre-loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF-A, -B, -C, and -D as well as PDGFRalpha and beta was detected. Depletion of PDGFRbeta in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT-PCR. PDGF-BB, but not PDGF-AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF-BB were eliminated by depletion of PDGFRbeta in MSCs except that PDGF-BB still suppressed osterix expression in PDGFRbeta-depleted MSCs. Depletion of PDGFRbeta significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRbeta gene deletion, we showed that PDGFRbeta signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRalpha on the osteogenic differentiation were very subtle. PDGFRbeta could represent an important target for guided tissue regeneration or tissue engineering of bone.

OBJECTIVE

CALGB80303 was a phase III trial of 602 patients with locally advanced or metastatic pancreatic cancer comparing gemcitabine/bevacizumab versus gemcitabine/placebo. The study found no benefit in any outcome from the addition of bevacizumab to gemcitabine. Blood samples were collected and multiple angiogenic factors were evaluated and then correlated with clinical outcome in general (prognostic markers) and with benefit specifically from bevacizumab treatment (predictive markers).

METHODS

Plasma samples were analyzed via a novel multiplex ELISA platform for 31 factors related to tumor growth, angiogenesis, and inflammation. Baseline values for these factors were correlated with overall survival (OS) using univariate Cox proportional hazard regression models and multivariable Cox regression models with leave-one-out cross validation. Predictive markers were identified using a treatment by marker interaction term in the Cox model.

RESULTS

Baseline plasma was available from 328 patients. Univariate prognostic markers for OS were identified including: Ang2, CRP, ICAM-1, IGFBP-1, TSP-2 (all P < 0.001). These prognostic factors were found to be highly significant, even after adjustment for known clinical factors. Additional modeling approaches yielded prognostic signatures from multivariable Cox regression. The gemcitabine/bevacizumab signature consisted of IGFBP-1, interleukin-6, PDGF-AA, PDGF-BB, TSP-2; whereas the gemcitabine/placebo signature consisted of CRP, IGFBP-1, PAI-1, PDGF-AA, P-selectin (both P < 0.0001). Finally, three potential predictive markers of bevacizumab efficacy were identified: VEGF-D (P < 0.01), SDF1 (P < 0.05), and Ang2 (P < 0.05).

CONCLUSIONS

This study identified strong prognostic markers for pancreatic cancer patients. Predictive marker analysis indicated that plasma levels of VEGF-D, Ang2, and SDF1 significantly predicted for benefit or lack of benefit from bevacizumab in this population.

Human platelet-derived growth factor (hPDGF) is likely to be important in stimulating tissue repair, based upon its in vivo chemotactic and stimulatory activities for inflammatory cells and fibroblasts and upon the presence of PDGF and related proteins in platelets, macrophages, and activated fibroblasts, cell types that make up the milieu of the healing wound. Recombinant human c-sis (rPDGF-B), homodimers of the B chain of PDGF, were compared with hPDGF in vitro. rPDGF-B was immunologically similar to hPDGF and, at identical concentrations, similar to hPDGF in stimulating fibroblast mitogenesis and chemotaxis of polymorphonuclear leukocytes, monocytes, and fibroblasts. Purified hPDGF and rPDGF-B were also tested in vivo for potency in a model of tissue repair using a linear incision wound through rat dermis. A single application of hPDGF or rPDGF-B (2-20 micrograms/wound) in a slow release vehicle at the time of wounding resulted in a dose-dependent, statistically highly significant increase of breaking strength of treated wounds. Wound healing in animals treated with rPDGF-B was 170% stronger and accelerated by 2 d during the first week over control wounds and by 4-6 d over the next 2 wk. Histologic evaluation of growth factor-treated wounds correlated the in vitro chemotactic activity and the accelerated healing of wounds with a striking inflammatory cell infiltrate early after wounding, markedly increased formation of granulation tissue by 4-d, and increased fibrosis by 14 d in comparison to control wounds. The results thus demonstrate that rPDGF-B is fully active in in vitro tests of mitogenesis and chemotaxis and, for the first time, demonstrate directly that PDGF significantly advances wound healing in incisional wounds of experimental animals.

Platelet-derived growth factors (PDGF) constitute a family of four gene products (PDGF-A-D) acting by means of two receptor tyrosine kinases, PDGFR alpha and beta. Three of the ligands (PDGF-A, -B, and -C) bind to PDGFR alpha with high affinity. Knockout of pdgf-a in mice has demonstrated a role for PDGF-A in the recruitment of smooth muscle cells to the alveolar sacs and their further compartmentalization into alveoli. Although this is a late, postnatal step in lung development, pdgf-a antisense oligonucleotides were previously shown to inhibit epithelial branching in rat lung explants in vitro, which reflects an early embryonic process. These conflicting results may be explained by substitution of genetic loss of pdgf-a by maternal transfer of PDGF-A to the knockout embryo or the presence of other PDGFR alpha agonists (PDGF-B and -C) in vivo, potentially masking an effect of PDGF-A on branching morphogenesis. Alternatively, the administration of pdgf-a antisense oligonucleotides affected other processes than the intended. To discriminate between these opposing possibilities, we have analyzed lung development in pdgfr alpha -/- embryos and lung primordia grown in vitro. Our analysis shows that, while the pdgfr alpha -/- lungs and explanted lung rudiments were smaller than normal, branching morphogenesis appears qualitatively intact and proceeds until at least embryonic day 15.5, generating both prospective conducting and respiratory airways. We conclude that, although PDGF-AA signaling over PDGFR alpha may have direct or indirect roles in overall lung growth, it does not specifically control early branching of the lung epithelium.

alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.

Smooth muscle cell (SMC) migration and proliferation and extracellular matrix remodeling are essential aspects of the arterial response to injury, vessel development, and atherogenesis. Matrix metalloproteinase (MMP) expression is associated with SMC proliferation and migration after arterial injury. To assess the role of MMPs in SMC proliferation and migration and intimal thickening, we measured the effect of the synthetic MMP inhibitor BB94 (Batimastat) on DNA synthesis and migration of SMCs in vitro as well as the formation of a neointima after balloon injury to the rat carotid artery. BB94 dose-dependently inhibited SMC migration induced by platelet-derived growth factor (PDGF)-BB through a filter coated with a thick basement membrane matrix (Matrigel) layer but did not show any inhibitory effect on SMC migration through a lightly coated filter. At concentrations up to 1 mumol/L, BB94 did not alter DNA synthesis induced by PDGF-AA or PDGF-BB. Treatment with 30 mg BB94.kg-1.d-1 IP for 7 or 14 days after balloon injury to the rat carotid artery decreased the total number of intimal SMC nuclei and suppressed intimal thickening. SMC proliferation (5-bromo-2'-deoxyuridine labeling) was decreased in the media at 2 days, whereas it was increased in the intima at 7 but not 14 days. These results suggest that BB94 inhibits intimal thickening after arterial injury by decreasing SMC migration and proliferation and support the conclusion that MMPs play a significant role in regulating intimal thickening in injured arteries.

The vitreous contains a plethora of growth factors that are strongly implicated in the formation of fibroproliferative diseases such as proliferative vitreoretinopathy. Although platelet-derived growth factors (PDGFs) are present in the vitreous, vitreal growth factors outside of the PDGF family activated the PDGF alpha receptor (PDGFRalpha) and promoted disease progression in a rabbit model of proliferative vitreoretinopathy (H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.) In this report we investigated the mechanism by which non-PDGFs activated PDGFRalpha. We found that non-PDGFs increased the cellular level of reactive oxygen species (ROS) and that this event was necessary and sufficient for phosphorylation of PDGFRalpha. We speculated that the underlying mechanism was ROS-mediated inhibition of phosphotyrosine phosphatases, which antagonize receptor auto-phosphorylation. However, this did not appear to be the case. Non-PDGFs promoted tyrosine phosphorylation of catalytically inactive PDGFRalpha, and thereby indicated that at least one additional tyrosine kinase was involved. Indeed, preventing expression or blocking the kinase activity of Src family kinases suppressed non-PDGF-dependent tyrosine phosphorylation of PDGFRalpha. Thus non-PDGFs increased the level of ROS, which activated Src family kinases and resulted in phosphorylation of PDGFRalpha. Finally, although non-PDGFs induced only modest phosphorylation of PDGFRalpha, proliferation and survival of cells in response to non-PDGFs was significantly enhanced by expression of PDGFRalpha. These studies reveal a novel mechanism for activation of PDGFRalpha that appears capable of enhancing the responsiveness of cells to growth factors outside of the PDGF family.

Evidence from clinical, animal and cell culture studies demonstrates that increased autotaxin (ATX) expression is responsible for enhancing tumor progression, cell migration, metastases, angiogenesis and chemo-resistance. These effects depend mainly on the rapid formation of lysophosphatidate (LPA) by ATX. Circulating LPA has a half-life of about 3 min in mice and it is degraded by the ecto-activities of lipid phosphate phosphatases (LPPs). These enzymes also hydrolyze extracellular sphingosine 1-phosphate (S1P), a potent signal for cell division, survival and angiogenesis. Many aggressive tumor cells express high ATX levels and low LPP activities. This favors the formation of locally high LPA and S1P concentrations. Furthermore, LPPs attenuate signaling downstream of the activation of G-protein coupled receptors and receptor tyrosine kinases. Therefore, we propose that the low expression of LPPs in many tumor cells makes them hypersensitive to growth promoting and survival signals that are provided by LPA, S1P, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). One of the key signaling pathways in this respect appears to be activation of phospholipase D (PLD) and phosphatidate (PA) production. This is required for the transactivations of the EGFR and PDGFR and also for LPA-induced cell migration. PA also increases the activities of ERK, mTOR, myc and sphingosine kinase-1 (SK-1), which provide individual signals for cells division, survival, chemo-resistance and angiogenesis. This review focuses on the balance of signaling by bioactive lipids including LPA, phosphatidylinositol 3,4,5-trisphosphate, PA and S1P versus the action of ceramides. We will discuss how these lipid mediators interact to produce an aggressive neoplastic phenotype.

We have investigated the regulation of mRNA levels for thrombospondin, a platelet-derived growth factor (PDGF)-regulated secreted glycoprotein, in cultures of rat vascular smooth muscle cells (SMC). A thrombospondin cDNA hybridizes to a single 5.5-kilobase SMC message which is greatly induced by serum or PDGF. When quiescent SMC are treated with PDGF, thrombospondin levels are induced rapidly (within 15 min) and in a dose-dependent manner. The induction of thrombospondin message levels parallels PDGF-mediated mitogenesis. PDGF-mediated increases in thrombospondin mRNA can be blocked in the presence of actinomycin D, suggesting that PDGF regulates the thrombospondin message at the level of gene transcription. In the presence of cycloheximide, PDGF "super-induces" the thrombospondin message. The data establish that mRNA levels for thrombospondin are regulated by PDGF in a manner similar to that of c-myc, c-fos, and other growth-regulatory gene products and extend our previous findings that thrombospondin secretion by SMC is dependent on exposure to PDGF. These observations are strongly suggestive of an important role for thrombospondin in the growth response of mesenchymal cells.

During vascular development, Notch signalling plays important roles in cell-cell communication and cell fate decisions. We studied expression of Notch 1-4 and its ligand Delta-like 4 (Dll4) in the developing retinal vasculature. Dll4 mRNA is strongly expressed in endothelial cells at the very tips of growing vessels ('tip cells') and also in arteries, where it is expressed in a segmented 'tiger's tail' pattern. This implies that developing retinal arteries contain different types of endothelial cells, Dll4-positive and Dll4-negative. The Dll4-positive stripes do not correspond to any obvious morphological property of the vascular network but correlate to some extent with the distribution of platelet derived growth factor B (PDGF-B) mRNA. However, PDGF-B expression is neither as artery-specific nor as clearly segmented as Dll4. Possible target cells for Dll4 signalling are retinal astrocytes (Notch1 positive), arterial pericytes (Notch3 positive) or arterial endothelial cells themselves (Notch4 positive). However, there is no clear reciprocity of Notch and Dll4 expression that allows identification of the interacting cells. Nevertheless, Dll4 stripes are a novel property of immature arteries, the origin and function of which remain to be explained.

1. Extracellular adenosine triphosphate (ATP) is mitogenic for vascular smooth muscle cells (VSMC) and stimulates several events that are important for cell proliferation: DNA synthesis, protein synthesis, increase of cell number, immediate early genes, cell-cycle progression, and tyrosine phosphorylation. 2. Receptor characterization indicates mitogenic effects of both P2U and P2Y receptors. The P2X receptor is lost in cultured VSMC and is not involved. Several related biological substances such as UTP, ITP, GTP, AP4A, ADP, and UDP are also mitogenic. 3. Signal transduction is mediated via Gq-proteins, phospholipase C beta, phospholipase D, diacyl glycerol, protein kinase C alpha, delta, Raf-1, MEK, and MAPK. 4. ATP acts synergistically with polypeptide growth factors (PDGF, bFGF, IGF-1, EGF, insulin) and growth factors acting via G-protein-coupled receptors (noradrenaline, neuropeptide Y, 5-hydroxytryptamine, angiotensin II, endothelin-1). 5. The mitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins. 6. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima-formation after angioplasty, and possibly hypertension.

Cardiac fibroblasts appear to be important in producing and maintaining the extracellular matrix (ECM) of the heart. The abnormal proliferation of cardiac fibroblasts and deposition of the ECM protein, collagen, associated with hypertension and myocardial infarction, may adversely affect the performance of the heart. Several groups of factors affect collagen gene expression and/or growth of cardiac fibroblasts. Angiotensin II, aldosterone and endothelins play a central role in the remodeling of the ECM in hypertension, and decrease collagenase activity and/or increase collagen synthesis in cultured cells. Regulatory peptides that are generally elevated at sites of injury, such as TGF-beta 1 and PDGF, increase collagen synthesis and/or stimulate mitogenesis. Mechanical stretch enhances collagen expression and cell proliferation, responses which could in part be due to integrin activation. Cytokines may stimulate or inhibit cell growth, the latter through prostaglandin formation. Angiotensin II is a principal determinant in vivo of cardiac fibroplasia and synthesis of the ECM proteins, collagen and fibronectin. Cardiac fibroblasts possess G-protein-coupled AT1 receptors for angiotensin II that couple to activation of multiple signalling pathways, including: phospholipase C-beta, with the subsequent release of Ca2+ from intracellular stores and activation of protein kinase C, mitogen-activated protein kinases, tyrosine kinases, phospholipase D, phosphatidic acid formation, and the STAT family of transcription factors. Cardiac fibroblasts respond to angiotensin II with hyperplastic/hypertrophic growth, and increased expression of collagen, fibronectin, and integrins. The mechanisms by which the AT1 receptor activates multiple signalling pathways are not known, although the receptor might interact at some level with both integrins and cytokine receptors. Different signalling pathways of the AT1 receptor may subserve different cellular responses, such as mitogenesis, ECM synthesis, or an inflammatory/stress response. Crosstalk among the signalling pathways of the AT1 receptor, and those of G-protein, cytokine, and growth-factor receptors, may determine the ultimate response of the cell.

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration.

CONCLUSIONS

CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.

Platelet-derived growth factor (PDGF) protein family members are potent mitogens and chemoattractants for mesenchymal cells. The classic PDGF ligands A and B are single-domain protein chains which are secreted as active dimers capable of activating their cognate PDGF receptors (PDGFRs). In contrast to PDGFs A and B, PDGF D contains an N-terminal complement subcomponent C1r/C1s, Uegf, and Bmp1 (CUB) domain and a C-terminal PDGF domain. PDGF D must undergo extracellular proteolytic processing, separating the CUB domain from the PDGF domain, before the PDGF domain can stimulate beta-PDGFR-mediated cell signal transduction. Here, we report that prostate carcinoma cells LNCaP and PC3 autoactivate latent full-length PDGF D into its active form under serum-independent conditions and that this autoactivation is inhibited by PAI-1, a urokinase plasminogen activator (uPA)/tissue plasminogen activator (tPA) inhibitor. Interestingly, uPA, but not the closely related protease tPA, is capable of processing recombinant latent PDGF DD into the active form. We identify the uPA cleavage site between the CUB and PDGF domains of the full-length PDGF D by mutational analysis and show that PDGF D and uPA colocalize in human prostate carcinoma. This evidence provides a direct link between uPA- and PDGF D-mediated cell signaling, which may contribute to the progression of prostate cancer.

Platelet-derived growth factors (PDGFs) were discovered almost two decades ago. The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well studied and they regulate a number of physiological and pathophysiological processes in many types of mesenchymal cells via two receptor tyrosine kinases, PDGF receptors alpha and beta. Recently, two additional PDGF polypeptide chains were discovered, namely PDGF-C and PDGF-D. The discovery of two additional ligands for the two PDGF receptors suggests that PDGF-mediated signaling is more complex than previously anticipated.

Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.

BACKGROUND

The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-alpha (PDGFR-alpha) and receptor-beta (PDGFR-beta) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-alpha and PDGFR-beta, respectively.

RESULTS

Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media.

CONCLUSIONS

These results indicate that PDGFR-beta plays a significant role in formation of fibrous atherosclerotic lesions and that regulation of the signal transduction through PDGFR-beta could affect atherogenesis in mice.

The purpose of this study was to determine the effect of the peroxisome proliferator-activated receptor gamma-(PPAR gamma) ligands troglitazone (TRO), rosiglitazone (RSG), and 15-deoxy-delta prostaglandin J2 (15d-PGJ2) on vascular smooth muscle cell (VSMC) migration directed by multiple chemoattractants. Involvement of mitogen-activated protein kinase (MAPK) in migration also was examined, because TRO was previously shown to inhibit nuclear events stimulated by this pathway during mitogenic signaling in VSMCs. Migration of rat aortic VSMCs was induced 5.4-fold by PDGF, 4.6-fold by thrombin, and 2.3-fold by insulin-like growth factor I (IGF-I; all values of p < 0.05). The PPAR gamma ligands 15d-PGJ2, RSG, or TRO all inhibited VSMC migration with the following order of potency: 15d-PGJ2>> RSG>> TRO. Inhibition of MAPK signaling with PDPDGF-, thrombin-, and IGF-I-induced migration. All chemoattractants induced MAPK activation. PPAR gamma ligands did not inhibit MAPK activation, suggesting a nuclear effect of these ligands downstream of MAPK. The importance of nuclear events was confirmed because actinomycin D also blocked migration. We conclude that PPAR gamma ligands are potent inhibitors of VSMC migration pathways, dependent on MAPK and nuclear events. PPAR gamma ligands act downstream of the cytoplasmic activation of MAPK and appear to exert their effects in the nucleus. Because VSMC migration plays an important role in the formation of atherosclerotic lesions and restenosis, PPAR gamma ligands like TRO and RSG, which ameliorate insulin resistance in humans, also may protect the vasculature from diabetes-enhanced injury.