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1.  PDGF mediates cardiac microvascular communication. 
Journal of Clinical Investigation  1998;102(4):837-843.
The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.
PMCID: PMC508947  PMID: 9710453
2.  PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors 
The Journal of Cell Biology  1994;125(4):917-928.
To delineate potential angiogenic roles of platelet-derived growth factor (PDGF), we have investigated PDGF and its receptors on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro (angiogenic endothelial cells). Initiation of cord/tube formation by angiogenic endothelial cells required bovine or human serum. Neutralization of PDGF-BB in human serum with a monoclonal anti-PDGF-BB antibody reduced cord/tube formation by 37 +/- 10%, whereas neutralizing anti-PDGF-AA and an IgG isotype-matched control antibody had no effect. DNA synthesis in response to PDGF-BB increased as the cords and tubes developed; furthermore, PDGF-BB induced the incorporation of BrdU in the nuclei of cells associated with these structures. PDGF beta-receptor (PDGF-beta) mRNA increased concomitantly with cord/tube formation, and PDGFR-beta were specifically localized by immunocytochemistry to developing and mature cords and tubes. However, PDGFR-beta transcripts and protein were undetectable in nonangiogenic endothelial cells, and PDGF alpha-receptor mRNA was not expressed in either endothelial cell strain. In contrast to nonangiogenic endothelial cells, angiogenic endothelial cells did not express the PDGF B-chain, the required ligand for the PDGFR-beta. We conclude that (a) PDGF-BB can contribute to angiogenesis in vitro, (b) PDGFR-beta are specific for cord/tube-forming endothelial cells and mediate endothelial proliferation and cord/tube formation, and (c) in angiogenic and nonangiogenic endothelial cells, the expression of PDGFR- beta and PDGF B-chain is inversely correlated. We therefore suggest that paracrine PDGF might amplify angiogenesis via direct action on endothelially expressed PDGFR-beta.
PMCID: PMC2120083  PMID: 7514607
3.  Functions of Paracrine PDGF Signaling in the Proangiogenic Tumor Stroma Revealed by Pharmacological Targeting  
PLoS Medicine  2008;5(1):e19.
Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.
Methods and Findings
We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.
This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.
Douglas Hanahan and colleagues investigate a paracrine regulatory circuit centered upon PDGF receptor signaling in cancer-associated fibroblasts and pericytes of a mouse model of cervical carcinogenesis.
Editors' Summary
Cancers—disorganized, life-threatening masses of cells—develop when cells acquire genetic changes that allow them to divide uncontrollably and to move into (invade) other tissues. Interactions with ostensibly normal cells in the tissue surrounding the tumor (the stroma) support the growth of these abnormal cells. The stroma contains endothelial cells and pericytes (which line the inside and coat the outside, respectively, of blood vessels), cancer-associated fibroblasts, and some immune system cells. Together, these cells support angiogenesis (the formation of a blood supply, which feeds the tumor), produce factors that stimulate tumor cell growth, and facilitate tumor cell invasion into surrounding tissues. One type of tumor with a prominent stromal compartment is cervical cancer. Precancerous changes in the epithelial cells lining the cervix (the structure that connects the womb to the vagina) are usually triggered by infection with human papillomavirus. Some of these early lesions, which are known as cervical intraepithelial neoplasias (CINs), develop into invasive cervical cancer, which is treated by surgery followed by chemotherapy or radiotherapy.
Why Was This Study Done?
The outlook for women whose cervical cancer is detected early is good but only 15%–30% of women whose cancer has spread out of the cervix survive for five years. If, as researchers believe, the stromal compartment is important in the development and growth (neoplastic progression) of cervical cancer, it might be possible to help these women by specifically targeting the cells in the stroma. However, relatively little is known about the role that the stroma plays in the neoplastic progression of cervical cancer or how it is regulated other than that a protein called platelet-derived growth factor (PDGF), which is made by the tumor cells, might be involved in its formation. In this study, the researchers have used a mouse model of cervical cancer (HPV/E2 mice) to investigate PDGF signaling in the tumor stroma. HPV/E2 mice develop CINs before they are three months old; by five months of age, 90% of them have invasive cervical cancer.
What Did the Researchers Do and Find?
The researchers report that PDGF was expressed in the cervixes of normal and HPV/E2 mice, mainly by epithelial cells, and that PDGF receptors (cell-surface proteins that bind PDGF and send a message into the cell that alters the expression of other proteins) were expressed on cells within normal stroma and in fibroblasts and pericytes in the stroma surrounding CINs and tumors (but not on the cancer cells). The expression of PDGF and its receptors increased slightly during tumor progression. Treatment of the HPV/E2 mice with imatinib, an inhibitor of PDGF signaling, slowed the progression of precancerous lesions, impaired the growth of invasive cancers, and reduced the number of blood vessels formed in the tumors and the coverage of these vessels with pericytes. Other experiments indicate that imatinib had these effects because its inhibition of stromal PDGF receptors suppressed the expression of FGF-7 (a factor that encourages epithelial cell division) and FGF-2 (a proangiogenic factor) by cancer-associated fibroblasts. Finally, as in HPV/E2 mice, FGF-2 and PDGF receptors were expressed in the stroma of human cervical cancers whereas PDGF was expressed in the cancer cells.
What Do These Findings Mean?
These findings suggest that PDGF receptor signaling in the stromal cells associated with cervical tumors in mice has a functional role during tumor progression. More specifically, they suggest that PDGF released by the tumor cells triggers PDGF signaling in the stromal cells, which increases the expression of factors that both directly and indirectly stimulate the growth of the tumor cells. Confirmation of this scheme will require additional experiments in mouse models of cervical cancer and the careful examination of more human material. Importantly, although approaches that work in mice do not always work in people, the current findings suggest that targeted therapeutics that prevent the stromal support of tumor growth (such as inhibitors of PDGF receptor signaling) might provide a complementary approach to conventional treatments that target the cancer cells themselves.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer, including information about cervical cancer (in English and Spanish)
The UK charity Cancerbackup also provides information on all aspects of cancer, including information on cervical cancer and on imatinib
Wikipedia has pages on platelet-derived growth factor, on PDGF receptors, and on imatinib (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2214790  PMID: 18232728
4.  Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells 
Diabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) were downregulated in ischemic limbs of diabetic mice, although the underlying mechanisms remained elusive. Protein kinase C (PKC) is a family of serine/threonine kinases and is known to be involved in angiogenesis. The purpose of this study is to elucidate the mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes.
Human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) cultured in normoglycemic or hyperglycemic conditions were examined. We also examined the effects of PKC inhibition on the PDGF-C/PDGFR-α axis in endothelial cells exposed to hyperglycemia.
Hyperglycemia inhibited proliferation and decreased viability of both HUVECs and HMVECs. Hyperglycemic endothelial cells exhibited decreased PDGFR-α expression both at messenger RNA (mRNA) and protein levels, while there was no significant change in expression of PDGF-C. We also found that expression of PKC-α, one of the PKC isoforms, was increased in hyperglycemic endothelial cells and that inhibition of PKC upregulated PDGFR-α expression in these cells. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by PDGF-C was significantly attenuated in hyperglycemic endothelial cells, whereas inhibition of PKC effectively reversed these inhibitory effects. Moreover, inhibition of PKC also promoted angiogenesis induced by PDGF-C in hyperglycemic endothelial cells, which was not observed in vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis.
These findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of hyperglycemia through upregulation of PKC. Targeting PKC to restore PDGF-C signaling might be a novel therapeutic strategy for the treatment of vascular complications in diabetes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-015-0180-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4334399
Platelet-derived growth factor C (PDGF-C); Diabetes; Therapeutic angiogenesis; Protein kinase C (PKC); Hyperglycemia; Endothelial cells
5.  Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells. 
Journal of Clinical Investigation  1997;100(11):2800-2809.
Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) regulate mesangial cell proliferation and matrix production in vitro and in vivo and crucially participate in the pathogenesis of glomerulonephritis. We investigated whether PDGF-BB and bFGF influence nitric oxide (NO) production, another important effector molecule in inflammatory glomerular injury. Inducible NO synthase (iNOS) induction in rat glomerular mesangial cells has been described in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1beta (IL-1beta) or elevation of cyclic AMP (cAMP). Treatment of mesangial cells with IL-1beta induces iNOS activity measured as nitrite levels in cell culture supernatants. Coincubation of mesangial cells with PDGF-BB inhibits production of nitrite by approximately 95%. This effect can be reversed by the simultaneous incubation of PDGF-BB in the presence of calphostin C, a potent and selective inhibitor of protein kinase C. In contrast, incubation of cells in the presence of bFGF potentiates IL-1beta-induced production of NO and is functionally associated with an increased rate of apoptosis of mesangial cells. Western blot analyses reveal that PDGF-BB causes a decrease in the formation of iNOS protein which is preceded by decreases in iNOS mRNA steady state levels. bFGF drastically increases iNOS protein levels as well as the corresponding iNOS mRNA steady state levels. Nuclear run-on experiments reveal that PDGF-BB decreases the IL-1beta-induced transcription rate of the iNOS gene, whereas bFGF potentiates the transcriptional activity of the iNOS gene. Northern blot analyses demonstrate that bFGF strongly potentiates the formation of IL-1beta-induced IL-1 type I receptor mRNA levels, whereas PDGF-BB has no effect. Treatment of mesangial cells with the membrane-permeable cAMP analogue N6, O-2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) markedly increases the production of nitrite. Whereas PDGF-BB does not affect cAMP-induced nitrite levels, bFGF strongly potentiates them. PDGF-BB alters neither cAMP-induced iNOS protein levels nor the corresponding iNOS mRNA steady state levels. By contrast, bFGF superinduces cAMP-stimulated iNOS protein and iNOS mRNA levels. These changes by bFGF are due to an increase in cAMP-induced transcriptional activity of the iNOS gene which is not affected by PDGF-BB. In summary, the results show that PDGF and bFGF differentially regulate iNOS expression in mesangial cells in a stimulus-specific way. The timely sequence of expression of PDGF and bFGF and of cytokines like IL-1 will crucially determine the amounts of NO produced and the functional consequences thereof in the course of progressive glomerular diseases.
PMCID: PMC508485  PMID: 9389745
6.  Chrysotile asbestos upregulates gene expression and production of alpha-receptors for platelet-derived growth factor (PDGF-AA) on rat lung fibroblasts. 
Journal of Clinical Investigation  1993;92(1):425-430.
PDGF isoforms have been postulated to serve as mediators of fibroblast proliferation and chemotaxis during lung fibrogenesis induced by asbestos inhalation. We have studied the interaction of chrysotile asbestos fibers with rat lung fibroblasts (RLF) in vitro and the consequent changes in PDGF receptor mRNA expression, PDGF binding, and mitogenic activity of PDGF isoforms. Northern blot analysis revealed that mRNA for the PDGF-receptor alpha subtype (PDGF-R alpha) on RLF was upregulated after a 24-h exposure to asbestos in culture (0.5-15 micrograms fibers/cm2). [125I]PDGF-BB receptor assays showed that normal RLF possess mainly PDGF-R beta and a paucity of PDGF-R alpha. In agreement with the Northern data, saturation binding of [125I]PDGF-BB to RLF exposed to asbestos demonstrated an approximately 40% increase in binding sites accompanied by a twofold decrease in receptor affinity. Treating asbestos-exposed RLF with PDGF-AA, which binds only PDGF-R alpha, blocked the PDGF binding sites that were upregulated by fiber exposure. PDGF-AA had increased mitogenic potency for fiber-exposed RLF, but PDGF-BB was a less potent mitogen for these RLF. Nonfibrogenic carbonyl iron spheres induced similar changes in PDGF growth responses. These data show that inorganic particulates alter the PDGF-R alpha population on RLF without significant change in PDGF-R beta.
PMCID: PMC293628  PMID: 8392089
7.  Expression of PDGF A-chain and beta-receptor genes during rat myoblast differentiation 
The Journal of Cell Biology  1990;110(5):1665-1672.
L6J1 rat myoblasts and rat skeletal muscle were studied for expression of mRNAs encoding PDGF A-chain, PDGF B-chain, PDGF alpha-receptor, and PDGF beta-receptor during in vitro and in vivo myoblast differentiation. RNA blot hybridizations demonstrated expression of the PDGF A-chain gene and the PDGF beta-receptor gene in L6J1 myoblasts and in crude muscle tissue isolated from developing rats. Transcripts of the PDGF A-chain were identified at all examined stages of in vitro and in vivo myogenic differentiation. Expression of the PDGF beta-receptor gene decreased in differentiated myotubes of L6J1 cells and in rat adult muscle tissue. Receptor binding assays demonstrated specific binding of PDGF-BB, but not -AA, to exponentially proliferating L6J1 myoblasts and to terminally differentiated L6J1 myotubes. The binding per cell nucleus was higher in exponentially proliferating myoblasts than in differentiated L6J1 myotubes. In serum free medium PDGF-BB was shown to increase c-fos protooncogene immunoreactivity in L6J1 myoblasts. In the presence of 0.5% FCS, PDGF-BB increased DNA synthesis in L6J1 myoblasts, while PDGF-AA showed no such effect. Differentiation, as monitored by myotube formation, was reduced in PDGF- BB-treated cultures. The possible role of PDGF in myoblast proliferation and differentiation is discussed.
PMCID: PMC2200189  PMID: 1970826
8.  Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing 
The Journal of Cell Biology  1988;107(5):1947-1957.
The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.
PMCID: PMC2115318  PMID: 2846589
9.  Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels 
Acta biomaterialia  2010;7(1):133-143.
The field of tissue engineering is severely limited by a lack of microvascularization in tissue engineered constructs. Biomimetic poly(ethylene glycol) hydrogels containing covalently immobilized platelet-derived growth factor BB (PDGF-BB) were developed to promote angiogenesis. Poly(ethylene glycol) hydrogels resist protein absorption and subsequent non-specific cell adhesion, thus providing a “blank slate”, which can be modified through the incorporation of cell adhesive ligands and growth factors. PDGF-BB is a key angiogenic protein able to support neovessel stabilization by inducing functional anastomoses and recruiting pericytes. Due to the widespread effects of PDGF in the body and a half-life of only 30 min in circulating blood, immobilization of PDGF-BB may be necessary. In this work bioactive, covalently immobilized PDGF-BB was shown to induce tubulogenesis on two-dimensional modified surfaces, migration in three-dimensional (3D) degradable hydrogels and angiogenesis in a mouse cornea micro-pocket angiogenesis assay. Covalently immobilized PDGF-BB was also used in combination with covalently immobilized fibroblast growth factor-2, which led to significantly increased endothelial cell migration in 3D degradable hydrogels compared with the presentation of each factor alone. When a co-culture of endothelial cells and mouse pericyte precursor 10T1/2 cells was seeded onto modified surfaces tubule formation was independent of surface modifications with covalently immobilized growth factors. Furthermore, the combination of soluble PDGF-BB and immobilized PDGF-BB induced a more robust vascular response compared with soluble PDGF-BB alone when implanted into an in vivo mouse cornea micropocket angiogenesis assay. Based on these results, we believe bioactive hydrogels can be tailored to improve the formation of functional microvasculature for tissue engineering.
PMCID: PMC3049810  PMID: 20801242
Angiogenesis; Hydrogel; Poly(ethylene glycol); Platelet-derived growth factor; Biofunctional materials
10.  Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant. 
Molecular and Cellular Biology  1993;13(7):4066-4076.
A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.
PMCID: PMC359956  PMID: 8321214
11.  Platelet-Derived Growth Factor Involvement in Myocardial Remodeling following Infarction 
Cardiac remodeling occurs in the infarcted heart (MI). The underlying regulatory mechanisms are under investigation. Platelet-derived growth factor (PDGF) is a family of growth factors that stimulates cell growth, differentiation and migration. Herein, we sought to determine whether PDGF is involved in cardiac repair/remodeling following MI.
Methods and Results
The temporal and spatial expression of PDGF isoforms (A, B, C and D) and PDGF receptor (PDGFR)-α and β as well as cell types expressing PDGF were examined in the infarcted rat heart. Sham-operated rats served as controls. We found that the normal myocardium expressed all PDGF isoforms, and cell types expressing PDGF were primarily interstitial cells. Following MI, PDGF-A and D were significantly increased in the infarcted myocardium during 6 weeks of the observation period and cells expressing PDGF-A and D were primarily endothelial cells, macrophages and myofibroblasts (myoFb). PDGF-B and C expression was, however, reduced in the infarcted heart. In the noninfarcted myocardium, PDGF-D expression was increased in the late stage of MI and cells expressing PDGF-D were predominantly fibroblasts. Both PDGFR-α and β were significantly increased in the infarcted myocardium in the early and late stages of MI and in the noninfarcted myocardium in the late stage of MI.
Enhanced PDGF-A, PDGF-D and PDGFR are coincident with angiogenesis, inflammatory and fibrogenic responses in the infarcted myocardium, suggesting their regulation on cardiac repair. Elevated PDGF-D in the noninfarcted myocardium suggests its involvement in the development of interstitial fibrosis that appears in the late stage of MI.
PMCID: PMC3628689  PMID: 21767547
Myocardial infarction; cardiac repair/remodeling; PDGF; PDGFR
12.  Malignant stroma increases luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth factor signaling 
BMC Cancer  2014;14(1):735.
Luminal, estrogen receptor-positive breast cancers represent more than 70% of cases. Despite initial good prognoses one third of Luminal cancers eventually recur locally or at distant sites and exhibit hormone resistance. Here we demonstrate that factors elaborated by malignant stromal cells can induce Luminal tumor cells proliferation and promote angiogenesis and hormone independence. We recently isolated a malignant mouse mammary gland stromal cell line named BJ3Z that increases proliferation and angiogenesis in estrogen-free xenografted Luminal MCF-7 breast cancer cells.
BJ3Z and Normal mouse mammary Fibroblasts (NMFs) were expression profiled using microarray assays. Messenger RNA levels were confirmed by RT-PCR and by immunohistochemistry (IHC). Breast cancer MCF-7, BT-474, BT-20 and MDA-MB-231cell lines and stromal BJ3Z and NMFs were grown for in vitro assays: breast cancer cell lines were treated with stromal cells conditioned media, for three-dimensional (3D) mono and co-cultures in Matrigel, proliferation was measured by Bromo-deoxyuridine (BrdU) incorporation using IHC. Tubule formation in vitro, a proxy for angiogenesis, was assessed using 3D cultured Human Umbilical cord Vascular Endothelial Cells (HUVEC).
We show that under estrogen-free conditions, BJ3Z cells but not NMFs increase proliferation of co-cultured Luminal but not basal-like human breast cancer cells in 2D or as 3D Matrigel colonies. Gene expression profiling, RT-PCR analysis and IHC of colony-derived BJ3Z cells and NMFs shows that Platelet Derived Growth Factor ligands (PDGF-A and -B) are elaborated by BJ3Z cells but not NMFs; while PDGF receptors are present on NMFs but not BJ3Z cells. As a result, in colony co-culture assays, BJ3Z cells but not NMFs increase MCF-7 cell proliferation. This can be mimicked by direct addition of PDGF-BB, and blocked by the PDGF receptor inhibitor Imatinib Mesylate. Both normal and malignant stromal cells enhance angiogenesis in an in vitro model. This effect is also due to PDGF and is suppressed by Imatinib.
We provide evidence that Luminal breast cancer cells can be targeted by the PDGF signaling pathway leading to estrogen-independent proliferation and angiogenesis. We speculate that stroma-directed therapies, including anti-PDGFR agents like Imatinib, may be useful in combination with other therapies for treatment of luminal cancers.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-735) contains supplementary material, which is available to authorized users.
PMCID: PMC4190420  PMID: 25274034
Stroma; Proliferation; Angiogenesis; Breast cancer
13.  SDF-1α induces PDGF- B expression and the differentiation of bone marrow cells into pericytes 
Molecular cancer research : MCR  2011;9(11):1462-1470.
Platelet derived growth factor B (PDGF-B) and its receptor, PDGFR-β, play a critical role in pericyte maturation; however, the mechanisms by which PDGF-B is up-regulated in the tumor microenvironment remain unclear. We previously demonstrated that up-regulating stromal-derived factor, SDF-1α, in vascular endothelial growth factor (VEGF165)-inhibited Ewing’s sarcoma tumors (TC/siVEGF7-1) induced PDGF-B mRNA expression, increased infiltration and differentiation of bone marrow cells (BMCs) into pericytes and, rescued tumor growth. The purpose of this study was to investigate the mechanism by which SDF-1α increased PDGF-B expression and the role of this pathway in BM-derived pericyte differentiation. We demonstrated that SDF-1α induced expression of PDGF-B mRNA and protein both in vitro and in vivo. In contrast, inhibiting SDF-1α down-regulated PDGF-B. We cloned the 2-kb pdgf-b promoter fragment and showed that SDF-1α activates PDGF-B via a transcriptional mechanism. Chromatin immunoprecipitation demonstrated that the ELK-1 transcription factor binds to the pdgf-b promoter in response to SDF-1α. We confirmed the correlation between the SDF-1α/PDGF-B pathway and the differentiation of PDGFR-β+ BMCs into mature pericytes using an in vitro assay. These findings demonstrate that SDF-1α regulates PDGF-B expression and that this regulation plays a critical role in the differentiation of PDGFR-β+ BMCs into mature pericytes.
PMCID: PMC3219839  PMID: 21911740
14.  Disruption of Platelet-Derived Growth Factor–Dependent Phosphatidylinositol 3-Kinase and Phospholipase Cγ 1 Activity Abolishes Vascular Smooth Muscle Cell Proliferation and Migration and Attenuates Neointima Formation In Vivo 
We tested the hypothesis whether selective blunting of platelet-derived growth factor (PDGF)–dependent vascular smooth muscle cell (VSMC) proliferation and migration is sufficient to prevent neointima formation after vascular injury.
To prevent neointima formation and stent thrombosis after coronary interventions, it is essential to inhibit VSMC proliferation and migration without harming endothelial cell function. The role of PDGF—a potent mitogen and chemoattractant for VSMC that does not affect endothelial cells—for neointima formation remains controversial.
To decipher the signaling pathways that control PDGF beta receptor (βPDGFR)–driven VSMC proliferation and migration, we characterized 2 panels of chimeric CSF1R/βPDGFR mutants in which the binding sites for βPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase [PI3K], GTPase activating protein of ras, SHP-2, phospholipase Cγ 1 [PLCγ]) were individually mutated. Based on in vitro results, the importance of PDGF-initiated signals for neointima formation was investigated in genetically modified mice.
Our results indicate that the chemotactic response to PDGF requires the activation of Src, PI3K, and PLCγ, whereas PDGF-dependent cell cycle progression is exclusively mediated by PI3K and PLCγ. These 2 signaling molecules contribute to signal relay of the βPDGFR by differentially regulating cyclin D1 and p27kip1. Blunting of βPDGFR-induced PI3K and PLCγ signaling by a combination mutant (F3) completely abolished the mitogenic and chemotactic response to PDGF. Disruption of PDGF-dependent PI3K and PLCγ signaling in mice expressing the F3 receptor led to a profound reduction of neointima formation after balloon injury.
Signaling by the activated βPDGFR, particularly through PI3K and PLCγ, is crucial for neointima formation after vascular injury. Disruption of these specific signaling pathways is sufficient to attenuate pathogenic processes such as vascular remodeling in vivo.
PMCID: PMC3732311  PMID: 21679854
PI-3 kinase; PLCγ; platelet-derived growth factor; proliferation; restenosis
15.  PDGF in gliomas: more than just a growth factor? 
Platelet-derived growth factor B (PDGF-B) is a growth factor promoting and regulating cell migration, proliferation, and differentiation, involved in both developmental processes and in maintaining tissue homeostasis under strict regulation. What are the implications of prolonged or uncontrolled growth factor signaling in vivo, and when does a growth factor such as PDGF-B become an oncogene? Under experimental conditions, PDGF-B induces proliferation and causes tumor induction. It is not known whether these tumors are strictly a PDGF-B-driven proliferation of cells or associated with secondary genetic events such as acquired mutations or methylation-mediated gene silencing promoting neoplasia. If PDGF-B-driven tumorigenesis was only cellular proliferation, associated changes in gene expression would thus be correlated with proliferation and not associated with secondary events involved in tumorigenesis and neoplastic transformation such as cycle delay, DNA damage response, and cell death. Changes in gene expression might be expected to be reversible, as is PDGF-B-driven proliferation under normal circumstances. Since PDGF signaling is involved in oligodendrocyte progenitor cell differentiation and maintenance, it is likely that PDGF-B stimulates proliferation of a pool of cells with that phenotype, and inhibition of PDGF-B signaling would result in reduced expression of oligodendrocyte-associated genes. More importantly, inhibition of PDGF signaling would be expected to result in reversion of genes induced by PDGF-B accompanied by a decrease in proliferation. However, if PDGF-B-driven tumorigenesis is more than simply a proliferation of cells, inhibition of PDGF signaling may not reverse gene expression or halt proliferation. These fundamental questions concerning PDGF-B as a potential oncogene have not been resolved.
PMCID: PMC3339541  PMID: 22376240
Glioma; growth factor; oncogene; oncogenic stress; PDGF-B
16.  Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta. 
Molecular and Cellular Biology  1994;14(10):6727-6735.
The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.
PMCID: PMC359203  PMID: 7935392
17.  A selective cyclic integrin antagonist blocks the integrin receptors αvβ3 and αvβ5 and inhibits retinal pigment epithelium cell attachment, migration and invasion 
BMC Ophthalmology  2005;5:16.
Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors αvβ3 and αvβ5, was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins αvβ3 and αvβ5 on RPE cells was examined.
The effect of a cyclic integrin antagonist and a control peptide (0.01 μg/ml to 300 μg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H3-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors αvβ3 and αvβ5 was evaluated by flow cytometry.
The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1–10 μg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3–10 μg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1–10 μg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3μg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors αvβ3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and αvβ5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold).
A selective inhibition of the integrin receptors αvβ3 and αvβ5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.
PMCID: PMC1184086  PMID: 15987521
18.  PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts 
Respiratory Research  2009;10(1):119.
Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).
Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.
The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.
During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.
PMCID: PMC2799395  PMID: 19939260
19.  Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rβ 
Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet-derived growth factor (PDGF)-BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF-Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF-BB. CSMC were enzymatically isolated from Sprague–Dawley rats, and the effect of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, transforming growth factor (TGF), IL-17A and IL-2 on CSMC growth and responsiveness to PDGF-BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid-induced colitis. Neither CM alone nor cytokines caused proliferation of early-passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF-BB. IL-1β, TNF-α and IL-17A, but not other cytokines, increased the effect of PDGF-BB because of up-regulation of mRNA and protein for PDGF-Rβ without change in receptor phosphorylation. PDGF-BB was identified in adult rat serum (RS) and RS-induced CSMC proliferation was inhibited by imatinib, suggesting that blood-derived PDGF-BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL-1β and TNF-α induced the early expression of PDGF-Rβ, while imatinib blocked subsequent RS-induced cell proliferation. Thus, pro-inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF-Rβ and serum-derived PDGF-BB, and control of PDGF-Rβ expression may be beneficial in chronic intestinal inflammation.
PMCID: PMC3955151  PMID: 24417820
TNBS; smooth muscle; mitosis; PDGF; phenotype; stricture; colitis
20.  Platelet-derived growth factor in glioblastoma—driver or biomarker? 
Upsala Journal of Medical Sciences  2014;119(4):298-305.
The platelet-derived growth factor (PDGF) family of mitogens exerts vital functions during embryonal development, e.g. in the central nervous system, where PDGF drives the proliferation of oligodendrocyte precursors. PDGF and PDGF receptors are co-expressed in human glioblastoma (GBM). Whether an aberrant activation of the PDGF receptor pathway is a driving force in glioma development has remained an open question. In experimental animals, overexpression of PDGF has convincingly been shown to induce tumors, both in wild-type animals (marmoset, rat, mouse) and in mice with targeted deletions of suppressor genes, e.g. Tp53 or Ink4A. Targeting the PDGF receptor in tumor-bearing mice leads to growth inhibition and reversion of the transformed phenotype. Findings of PDGF receptor amplification or mutations in human GBM are strong indicators of a causative role of the PDGF receptor pathway. However, clinical trials using PDGF receptor antagonists have been disappointing. In conclusion, a PDGF receptor profile may be a biomarker for a subgroup of GBM originating from a PDGF receptor-responsive cell. Although compelling experimental and clinical evidence supports the notion that the PDGF receptor pathway is a driver in GBM, formal proof is still missing.
PMCID: PMC4248069  PMID: 25342206
Brain tumors; molecular biology; oncogenes; tumor biology
21.  Effect of Sustained Gene Delivery of Platelet-Derived Growth Factor or Its Antagonist (PDGF-1308) on Tissue-Engineered Cementum 
Journal of periodontology  2004;75(3):429-440.
Cementum, a mineralized tissue lining the tooth root surface, is destroyed during the inflammatory process of periodontitis. Restoration of functional cementum is considered a criterion for successful regeneration of periodontal tissues, including formation of periodontal ligament, cementum, and alveolar bone. Short-term administration of platelet-derived growth factor (PDGF) has been shown to partially regenerate periodontal structures. Nonetheless, the role of PDGF in cementogenesis is not well understood. The aim of the present study was to determine the effect of sustained PDGF gene transfer on cementum formation in an ex vivo ectopic biomineralization model.
Osteocalcin (OC) promoter-driven SV40 transgenic mice were used to obtain immortalized cementoblasts (OCCM). The OCCM cells were transduced with adenoviruses (Ad) encoding either PDGF-A, an antagonist of PDGF signaling (PDGF-1308), a control virus (green fluorescent protein, GFP), or no treatment (NT). The transduced cells were incorporated into polymer scaffolds and implanted subcutaneously into severe combined immunodeficient (SCID) mice. The implants were harvested at 3 and 6 weeks for histomorphometric analysis of the newly formed mineralized tissues. Northern blot analysis was performed to determine the expression levels of mineral-associated genes including bone sialoprotein (BSP), OC, and osteopontin (OPN) in the cell-implant specimens at 3 and 6 weeks.
The results indicated mineralization was significantly reduced in both the Ad/PDGF-A and Ad/PDGF-1308 treated specimens when compared to the NT or Ad/GFP groups at 3 and 6 weeks (P <0.01). In addition, the size of the implants treated with Ad/PDGF-A and Ad/PDGF-1308 was significantly reduced compared to implants from Ad/GFP and NT groups at 3 weeks (P <0.05). At 6 weeks, the size of implants and mineral formation increased in NT, Ad/GFP, and Ad/PDGF-A groups, while the Ad/PDGF-1308 treated implants continued to decrease in size and mineral formation (P <0.01). Northern blot analysis revealed that in the Ad/PDGF-A treated implants OPN was increased, whereas OC gene expression was downregulated at 3 weeks. In the Ad/PDGF-1308 treated implants, BSP, OC, and OPN were all downregulated at 3 weeks. At 3 weeks, the Ad/PDGF-A treated implants contained significantly higher multinucleated giant cell (MNGC) density compared to NT, Ad/GFP, and Ad/PDGF-1308 specimens. The MNGC density in NT, Ad/GFP, and Ad/PDGF-A treated groups reduced over time, while the Ad/PDGF-1308 transduced implants continued to exhibit significantly higher MNGC density compared with the other treatment groups at 6 weeks.
The results showed that continuous exposure to PDGF-A had an inhibitory effect on cementogenesis, possibly via the upregulation of OPN and subsequent enhancement of MNGCs at 3 weeks. On the other hand, Ad/PDGF-1308 inhibited mineralization of tissue-engineered cementum possibly due to the observed downregulation of BSP and OC and a persistence of stimulation of MNGCs. These findings suggest that continuous exogenous delivery of PDGF-A may delay mineral formation induced by cementoblasts, while PDGF is clearly required for mineral neogenesis.
PMCID: PMC2596891  PMID: 15088882
Cementogenesis; dental cementum; growth factors; platelet-derived; protein; green fluorescent
22.  Stimulation of beta1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF beta-receptors 
The Journal of Cell Biology  1996;132(4):741-752.
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.
PMCID: PMC2199872  PMID: 8647902
23.  Regional expression of the platelet-derived growth factor and its receptors in a primate graft model of vessel wall assembly. 
Journal of Clinical Investigation  1993;92(1):338-348.
Healing baboon polytetrafluoroethylene grafts express PDGF mRNA in the neointima. Perfusates of graft segments also contain PDGF-like mitogenic activity. To extend these findings, we studied the expression and regional distribution of the PDGF protein isoforms and their receptors in this prosthetic graft model. By immunohistochemistry, as well as ELISA and Western blot analysis of tissue extracts, both PDGF-A and PDGF-B were identified in macrophages within the interstices of the synthetic material. In contrast, the neointima contained predominantly PDGF-A localized to the endothelial surface and the immediate subjacent smooth muscle cell layers. Tissue extracts of neointima and graft material were mitogenic for baboon aortic smooth muscle cells in culture; nearly all of this proliferative activity was blocked by a neutralizing anti-PDGF antibody. PDGF receptor beta-subunit mRNA and protein were easily detectable in the neointima and graft material. PDGF receptor alpha-subunit mRNA was also observed in the graft matrix and at lower levels in the neointima. This pattern of ligand and receptor expression further implicates locally produced PDGF as a regulator of neointimal smooth muscle cell growth in this model. The coexpression of ligand and receptor in the macrophage-rich matrix also suggests that PDGF may participate in the foreign body response.
PMCID: PMC293606  PMID: 8326002
24.  A Potential Oncogenic Activity of Platelet-Derived Growth Factor D in Prostate Cancer Progression 
Cancer research  2004;64(5):1722-1729.
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 NH2-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 β-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.
PMCID: PMC4171134  PMID: 14996732
25.  Differential Tumorigenic Potential and Matriptase Activation between PDGF B versus PDGF D in Prostate Cancer 
Molecular cancer research : MCR  2012;10(8):1087-1097.
The platelet-derived growth factors (PDGF A, B, C, and D) and their receptors (α-PDGFR and β-PDGFR) play an indispensible role in physiologic and pathologic conditions, including tumorigenesis. The transformative β-PDGFR is overexpressed and activated during prostate cancer progression, but the identification and functional significance of its complementary ligand have not been elucidated. This study examined potential oncogenic functions of β-PDGFR ligands PDGF B and PDGF D, using nonmalignant prostate epithelial cells engineered to overexpress these ligands. In our models, PDGF D induced cell migration and invasion more effectively than PDGF B in vitro. Importantly, PDGF D supported prostate epithelial cell tumorigenesis in vivo and showed increased tumor angiogenesis compared with PDGF B. Autocrine signaling analysis of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways found PDGF D–specific activation of the c-jun-NH2-kinase (JNK) signaling cascade. Using short hairpin RNA and pharmacologic inhibitors, we showed that PDGFD-mediated phenotypic transformation is β-PDGFR and JNK dependent. Importantly, we made a novel finding of PDGF D–specific increase in the shedding and activation of the serine protease matriptase in prostate epithelial cells. Our study, for the first time to our knowledge, showed ligand-specific β-PDGFR signaling as well as PDGF D–specific regulation of matriptase activity and its spatial distribution through shedding. Taken together with our previous finding that matriptase is a proteolytic activator of PDGF D, this study provides a molecular insight into signal amplification of the proteolytic network and PDGF signaling loop during cancer progression.
PMCID: PMC3929081  PMID: 22689130

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