Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.

The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts.

A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of platelet-derived growth factor B chain (PDGF-BB) and vascular endothelial growth factor 165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. Ten, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared with 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle alpha-actin (10%) and neuron-glia antigen 2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels. Disclosure of potential conflicts of interest is found at the end of this article.

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.

Chronic pulmonary hypertension leads to structural alterations of the lung vessels. The pathophysiology of this remodelling process is still poorly understood. Furthermore, the structural damage of the lung vessels limits the clinical success of vasodilator treatment. Given a genetic susceptibility, shear stress and inflammation are the principal pathogenetic factors involved in lung vessel remodelling. In this overview, we compared the lung vascular histology of patients with unexplained pulmonary hypertension, scleroderma, or acquired immune deficiency syndrome (AIDS)-associated pulmonary hypertension. We demonstrate the presence of inflammatory cells (T- and B-lymphocytes and macrophages) in plexiform lesions and discuss the potential role of growth factors (platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) in pulmonary hypertensive remodelling. Ultimately, we need to develop an in-depth understanding of the key mechanisms of gene expression and signalling pathways, which transduce signals generated from increased chronic shear stress (or from chronic hypoxia) and lead to vascular injury and repair.

OBJECTIVE

Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor beta subunit in a rat model of chronic cholestasis.

METHODS

Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor beta subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver.

RESULTS

In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells.

CONCLUSIONS

These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/vessel growth associated with the disease progression. However, target specificity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFRbeta) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFRbeta kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangiogenic factors, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFRbeta/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFRbeta/c-Src TKs in MM, providing a framework for future clinical trials.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.

Three recombinant homodimeric isoforms of platelet-derived growth factor (PDGF) were produced and purified in milligram quantities by expression of PDGF A- and B-chains in yeast cells. Structural analysis of the purified short and long variants of PDGF-AA (PDGF-AAS and PDGF-AAL) and PDGF-BB showed that they had been properly processed and assembled into dimers. PDGF-AAS and PDGF-AAL were found to bind only to the PDGF A-type receptor on human fibroblasts, with affinities of 0.1 and 0.2 nM, respectively. PDGF-BB bound to cells with A- and B-type receptors and to cells with B-type receptor only with affinities of 0.6 nM in both cases. Each fibroblast appeared to express about 4-5 times more B-type receptors than A-type receptors. The maximal mitogenic response to PDGF-BB of human fibroblasts was almost 2-fold higher than that induced by either of the two PDGF-AA forms. The three isoforms of PDGF also stimulated growth in soft agar of human fibroblasts with PDGF-BB inducing a higher maximal response.

Platelet-derived growth factor (PDGF) is a basic protein of relative molecular mass 30,000 (Mr 30K) composed of two polypeptide chains, designated PDGF A and PDGF B. The B-chain is encoded by the c-sis gene, the cellular counterpart of the simian sarcoma virus transforming gene v-sis. The PDGF A-chain cDNA clones recently isolated and sequenced from a transformed human clonal glioma cell line represent at least two alternatively spliced transcript species differing by 69 base pairs at the C-terminus. Here we demonstrate that the normal human umbilical vein endothelial cell (EC) A chain precursor lacks the 15 carboxy-terminal, highly basic amino acids encoded by the larger tumour cell cDNA. Surprisingly, culture media from monkey kidney cells (COS) transfected with the endothelial cDNA clone contained much less mitogenic activity than media from cells transfected with the longer tumour cell-derived A-chain cDNA. This functional difference appeared to be due to inefficient assembly or secretion of the recombinant endothelial-type growth factor. This suggests that some transformed cells may use alternative RNA splicing to modify normal growth factors and by so doing increase the efficiency of mitogen assembly or secretion.

OBJECTIVE

The purpose of this study was to examine expression of platelet-derived growth factor (PDGF) and PDGF receptors in the human cornea and to study the effects of the PDGF isotypes on proliferation and chemotaxis of human corneal fibroblasts. The effects of interleukin (IL)-1alpha, bone morphogenic protein (BMP)2, and BMP4 on chemotaxis of human corneal fibroblasts were also studied.

METHODS

mRNA expression was monitored with reverse transcription-polymerase chain reaction (RT-PCR) in primary cultured cells. Protein expression in fresh-frozen human corneal sections was studied with immunocytology. Chemotaxis was measured using a modified Boyden chamber, and proliferation was quantitated by cell counting.

RESULTS

PDGF A, PDGF B, PDGF receptor alpha, and PDGF receptor beta mRNAs were detected in corneal epithelial cells, fibroblasts, and endothelial cells in culture. The proteins were expressed in each major cell type in human corneal sections, with PDGF A and PDGF B detected at high levels in the epithelial basement membrane. PDGF, BMP2, and BMP4 had attractive chemotactic effects on corneal fibroblasts, with the PDGF BB dimer having a significantly greater positive chemotactic effect than the other PDGF isotypes. Interleukin-1alpha had a repulsive chemotactic effect on corneal fibroblasts. PDGF AA, AB, and BB stimulated proliferation of human corneal fibroblasts.

CONCLUSIONS

The PDGF growth factor receptor system is expressed in the human cornea. PDGF, BMP2, BMP4, and IL-1alpha may modulate keratocyte chemotaxis and proliferation during homeostasis and wound healing.

The signaling activity of Platelet-derived growth factors A and B (PDGF-A and PDGF-B) that is mediated through the two receptor kinases, PDGFR-alpha and PDGFR-beta has been shown to be critical for the development of the cardiovascular organs, the kidney, the lung and the central nervous system. During the cloning of genes for VEGF related proteins, we isolated a mouse cDNA that can encode for a protein of 345 amino acids. A comparison of the amino acid sequence reveals that this predicted gene product displays 95% identity to human PDGF-C. The mouse Pdgfc gene maps to a region of chromosome 17 that is syntenic to human chromosome 6p21.3 In E9. 5-E15.5 mouse embryo, Pdgfc is widely expressed in the surface ectoderm and later in the germinal layer of the skin, the olfactory and otic placode and their derivatives and the lining of the oral cavity. In the gut and visceral organs, such as the lung and the kidney, Pdgfc mRNA is first expressed in the endodermal epithelium and later in mesenchymal tissues associated with the endodermal structures. Similar to other PDGFs, Pdgfc is widely expressed in mesenchymal precursors and the myoblast of the smooth and skeletal muscles. Contrary to PDGF-A, Pdgfc is not expressed in the central nervous system, except in the cerebellum, and neurogenic derivatives of the neural crest cells. Pdgfc is also absent from the heart and the vascular endothelium

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

OBJECTIVE

The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines.

METHODS

Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively.

RESULTS

PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance.

CONCLUSIONS

These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.

Bone marrow-derived mesenchymal stem cells (MSCs) have been shown to localize to gliomas and deliver therapeutic agents. However, the clinical translation of MSCs remains poorly defined because previous studies relied on glioma models with uncertain relevance to human disease, typically xenograft models in immunocompromised mice. To address this shortcoming, we used the RCAS/Ntv-a system, in which endogenous gliomas that recapitulate the tumor and stromal features of human gliomas develop in immunocompetent mice. MSCs were harvested from bone marrow of Ntv-a mice and injected into the carotid artery of Ntv-a mice previously inoculated with RCAS-PDGF-B and RCAS-IGFBP2 to induce malignant gliomas (n = 9). MSCs were labeled with luciferase for in vivo bioluminescence imaging (BLI). After intra-arterial injection, BLI revealed MSCs in the right frontal lobe in seven of nine mice. At necropsy, gliomas were detected within the right frontal lobe in all these mice, correlating with the location of the MSCs. In the two mice without MSCs based on BLI, no tumor was found, indicating that MSC localization was tumor specific. In another cohort of mice (n = 9), MSCs were labeled with SP-DiI, a fluorescent vital dye. After intra-arterial injection, fluorescence microscopy revealed SP-DiI-labeled MSCs throughout tumors 1 to 7 days after injection but not in nontumoral areas of the brain. MSCs injected intravenously did not localize to tumors (n = 12). We conclude that syngeneic MSCs are capable of homing to endogenous gliomas in immunocompetent mice. These findings support the use of MSCs as tumor-specific delivery vehicles for treating gliomas.

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.

Mast cells are traditionally viewed as effector cells of immediate type hypersensitivity reactions. There is, however, a growing body of evidence that the cells might play an important role in the maintenance of tissue homeostasis and repair. We here present our own data and those from the literature elucidating the possible role of mast cells during wound healing. Studies on the fate of mast cells in scars of varying ages suggest that these cells degranulate during wounding, with a marked decrease of chymase-positive cells, although the total number of cells does not decrease, based on SCF-receptor staining. Mast cells contain a plethora of preformed mediators like heparin, histamine, tryptase, chymase, VEGF and TNF-alpha which, on release during the initial stages of wound healing, affect bleeding and subsequent coagulation and acute inflammation. Various additional vasoactive and chemotactic, rapidly generated mediators (C3a, C5a, LTB4, LTC4, PAF) will contribute to these processes, whereas mast cell-derived proinflammatory and growth promoting peptide mediators (VEGF, FGF-2, PDGF, TGF-beta, NGF, IL-4, IL-8) contribute to neoangiogenesis, fibrinogenesis or re-epithelization during the repair process. The increasing number of tryptase-positive mast cells in older scars suggest that these cells continue to be exposed to specific chemotactic, growth- and differentiation-promoting factors throughout the process of tissue remodelling. All these data indicate that mast cells contribute in a major way to wound healing. their role as potential initiators of or as contributors to this process, compared to other cell types, will however have to be further elucidated.

Digital clubbing is associated with many unrelated serious diseases but its pathogenesis remains a clinical enigma. It has been hypothesized that platelet clusters impacting in the distal vasculature mediate the morphological changes of clubbing. Since the multifunctional cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are released on platelet aggregation and are hypoxically regulated, the present study has examined their role in clubbing using immunohistochemistry. Basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-betaPDGF (p = 0.017), and HIF-1alpha and HIF-2alpha (p = 0.004 and p = 0.004, respectively) expression together with a significant increase in microvessel density (p = 0.03) in the stroma in clubbed digits compared with controls. There was no difference in CAIX (p = 0.25), TGF-betaPDGF are released after platelet impaction and that their expression is hypoxically enhanced in the stroma after capillary occlusion. VEGF may synergize with PDGF in inducing the stromal and vascular changes present in digital clubbing.

Phosphatidylinositol (PI) 3-kinase signaling regulates numerous cellular processes, including proliferation, migration, and survival, which are required for neointimal hyperplasia and restenosis. The effectors of PI 3-kinase are activated by the phospholipid products of PI 3-kinase. In this report, we investigated the hypothesis that overexpression of the tumor suppressor protein PTEN, an inositol phosphatase specific for the products of PI 3-kinase, would inhibit the vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia and restenosis. Effects of PTEN were assessed in primary rabbit VSMCs after overexpression with a recombinant adenovirus and compared with uninfected or control virus-infected cells. PTEN was expressed endogenously in VSMCs, and PTEN overexpression inhibited PDGF-induced phosphorylation of p70(s6k), Akt, and glycogen synthase kinase-3-alpha and -beta but not ERK1 or -2. Overexpression of PTEN significantly inhibited both basal and PDGF-mediated VSMC proliferation and migration, the latter possibly due in part to downregulation of focal adhesion kinase. Moreover, PTEN overexpression induced cleavage of caspase-3 and significantly increased apoptosis compared with control cells. Taken together, these results demonstrate that PTEN overexpression potently inhibits the VSMC responses required for neointimal hyperplasia and restenosis. Adenovirus-expressed PTEN may therefore provide a useful tool for the local treatment of these and other vascular proliferative disorders.

OBJECTIVE

Rapid induction of β-PDGF receptor (β-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of β-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent β-PDGFR activity, and assessed its prognostic implications in human cirrhosis.

METHODS

The impact of either loss or constitutive activation of β-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked β-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis.

RESULTS

Depletion of β-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-κB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of β-PDGFR in stellate cells. In the human cohort, the β-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis.

CONCLUSIONS

β-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Transforming growth factor beta 1 (TGF-beta 1) and its closely related homologue, TGF-beta 2, rapidly induce growth factor gene expression by freshly isolated human peripheral blood monocytes. Within 3 h of exposure to TGF-beta, mRNA species specific for interleukin-1 (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) were observed. By 14-18 h, cytokine bioactivity and protein were detected in the culture supernatants. Furthermore, not only TGF-beta 1, but also TGF-beta 2 mRNA are expressed constitutively in unstimulated monocytes. However, in response to exogenous TGF-beta (beta 1 or beta 2), only TGF-beta 1 gene expression is upregulated, and the expression of TGF-beta 2 mRNA is unchanged. This selective autoinduction of TGF-beta 1 appears to be controlled at both transcriptional and post-transcriptional levels. These paracrine and autocrine activities of TGF-beta suggest potential mechanisms through which an inflammatory response can be initiated and amplified. In addition, the TGF-beta enhancement of growth factor generation may promote fibrosis and angiogenesis relevant to physiological tissue repair as well as pathological fibrotic sequelae.

We recently established that the elastin-binding protein, which is identical to the spliced variant of beta-galactosidase, forms a cell surface-targeted complex with two proteins considered "classic lysosomal enzymes": protective protein/cathepsin A and neuraminidase-1 (Neu1). We also found that cell surface-residing Neu1 can desialylate neighboring microfibrillar glycoproteins and facilitate the deposition of insoluble elastin, which contributes to the maintenance of cellular quiescence. Here we provide evidence that cell surface-residing Neu1 contributes to a novel mechanism that limits cellular proliferation by desialylating cell membrane-residing sialoglycoproteins that directly propagate mitogenic signals. We demonstrated that treatment of cultured human aortic smooth muscle cells (SMCs) with either a sialidase inhibitor or an antibody that blocks Neu1 activity induced significant up-regulation in SMC proliferation in response to fetal bovine serum. Conversely, treatment with Clostridium perfringens neuraminidase (which is highly homologous to Neu1) decreased SMC proliferation, even in cultures that did not deposit elastin. Further, we found that pretreatment of aortic SMCs with exogenous neuraminidase abolished their mitogenic responses to recombinant platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-2 and that sialidosis fibroblasts (which are exclusively deficient in Neu1) were more responsive to PDGF-BB and IGF-2 compared with normal fibroblasts. Furthermore, we provide direct evidence that neuraminidase caused the desialylation of both PDGF and IGF-1 receptors and diminished the intracellular signals induced by the mitogenic ligands PDGF-BB and IGF-2.

Dermatofibrosarcoma protuberans (DFSP) is an aggressive spindle cell neoplasm. It is associated with the chromosomal translocation, t(17:22), which fuses the COL1A1 and PDGFbeta genes. We determined the characteristic gene expression profile of DFSP and characterized DNA copy number changes in DFSP by array-based comparative genomic hybridization (array CGH). Fresh frozen and formalin-fixed, paraffin-embedded samples of DFSP were analyzed by array CGH (four cases) and DNA microarray analysis of global gene expression (nine cases). The nine DFSPs were readily distinguished from 27 other diverse soft tissue tumors based on their gene expression patterns. Genes characteristically expressed in the DFSPs included PDGF beta and its receptor, PDGFRB, APOD, MEOX1, PLA2R, and PRKCA. Array CGH of DNA extracted either from frozen tumor samples or from paraffin blocks yielded equivalent results. Large areas of chromosomes 17q and 22q, bounded by COL1A1 and PDGF beta, respectively, were amplified in DFSP. Expression of genes in the amplified regions was significantly elevated. Our data shows that: 1) DFSP has a distinctive gene expression profile; 2) array CGH can be applied successfully to frozen or formalin-fixed, paraffin-embedded tumor samples; 3) a characteristic amplification of sequences from chromosomes 17q and 22q, demarcated by the COL1A1 and PDGF beta genes, respectively, was associated with elevated expression of the amplified genes.

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.