NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55-243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.

The primary objective of this study was to assess the safety of recombinant human (rh) platelet-derived growth factor-BB (PDGF-BB) and (rh) insulin-like growth factor-I (IGF-I) when applied to periodontal osseous defects in humans; a secondary objective was to begin to accrue data on the therapeutic dose of these growth factors (GFs) required to stimulate periodontal regeneration. Thirty-eight human subjects possessing bilateral osseous periodontal lesions were assigned to one of two treatment groups in a split-mouth design. Following full-thickness flap reflection, test sites received local application of the therapeutic drug delivered in coded syringes by a "masked" investigator. Two dose levels were tested, 50 micrograms/ml each of rhPDGF-BB and rhIGF-I in a gel vehicle (LD-PDGF/IGF-I) and 150 micrograms/ml each of rhPDGF-BB and rhIGF-I plus vehicle (HD-PDGF/IGF-I). Control treatment consisted of either conventional periodontal flap surgery or surgery plus vehicle. Safety analyses included physical examination, hematology, serum chemistry, urinalysis, antibody titers, and radiographic evaluation of bony changes. The primary therapeutic assessment was bone fill measured at re-entry 6 to 9 months after treatment. No local or systemic safety issues were found as a result of GF administration. No patients developed antibodies to the rhGF proteins. In subjects treated with LD-PDGF/IGF-I, there were no enhancements in periodontal regeneration compared to controls. However, in patients treated with HD-PDGF/IGF-I, statistically significant increases in alveolar bone formation were noted as measured by surgical re-entry 9 months following drug delivery (P < 0.05). This corresponded to an increase of 2.08 mm of new vertical bone height and 42.3% osseous defect fill in the HD-PDGF/IGF-I subjects versus only 0.75 mm and 18.5% gains in new bone height and osseous fill, respectively, in the controls. Furcation lesions, although limited in number, responded most favorably to treatment, with 2.8 mm horizontal osseous fill. The results from this study suggest that the local application of rhPDGF-BB and rhIGF-I to periodontal lesions is safe at the dose levels studied. LD-PDGF/IGF-I did not elicit increased defect fill compared to the control; however, HD-PDGF/IGF-I resulted in a significant promotion in bone regeneration. Additional studies are warranted to more fully characterize the effects of PDGF/IGF-I on periodontal regeneration in humans.

We have developed a panel of monoclonal antibodies to platelet-derived growth factor (PDGF) which have variable specificities for the three dimeric forms of the molecule (AA, AB, and BB). We have used these antibodies to detect and immunoaffinity purify the individual dimers from human platelet rich plasma. Extracts of outdated platelet preparations were initially chromatographed over CM-Sepharose and then passed over the Sepharose-coupled monoclonal antibodies in series in selectively isolate the three dimeric forms of PDGF. The PDGF eluted from the affinity columns was subsequently further purified by reversed-phase HPLC. From 300 units of outdated platelet preparations, we purified 58 micrograms of PDGF-BB and 140 micrograms of PDGF-AB. Using the monoclonal antibodies to develop PDGF dimer-specific ELISAs, it was observed that all three PDGF dimer forms are present in fresh human platelet extracts and that the ratios of the three dimer forms vary depending upon the extraction conditions used. The identification of all three PDGF dimer forms in human platelets point to the need to view PDGF isolated from human platelets by conventional techniques as a mixture of all three forms and not solely as PDGF-AB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.

Migration of chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow-derived mesenchymal stem cells in Boyden chamber assays. In human articular chondrocytes serum induced dose- and time-dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging-related decline in the ability of chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF-1, IL-8, BMP-4, and BMP-7. In summary, these results demonstrate that directed cell migration is inducible in human articular chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering.

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

Human platelet-derived growth factor (PDGF) occurs as three isoforms which are made up of disulfide-bonded A and B chains. The isoforms bind with different affinities to two different but structurally related cell surface receptors. The A type receptor binds all three isoforms (PDGF-AA, PDGF-AB, PDGF-BB) with high affinity, whereas the B type receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity but does not appear to bind PDGF-AA. We have utilized the differential effects of the three isoforms on actin reorganization and membrane ruffling in human foreskin fibroblasts to probe the idea that ligand-induced receptor dimerization is associated with receptor activation. Actin reorganization was found to be induced only by PDGF-AB and PDGF-BB and is therefore likely to be mediated by the B type receptor. Simultaneous addition of PDGF-AA, or downregulation of the A type receptor blocked the effect of PDGF-AB but not that of PDGF-BB. This is compatible with a model by which PDGF-AB binds to and dimerizes one A and one B type receptor; PDGF-AB therefore requires A type receptors in order to be functionally active at physiological concentrations. In cells with down-regulated A type receptors, high concentrations of PDGF-AB inhibited the effect of PDGF-BB on actin reorganization. We believe that this is due to a monovalent binding of PDGF-AB to the B type receptors which prevents PDGF-BB from dimerizing the receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another.

METHODS

Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles.

RESULTS

SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold).

CONCLUSIONS

The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

OBJECTIVE

Aberrant vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to the development of vascular pathologies, such as atherosclerosis and restenosis. MicroRNAs have recently emerged as critical modulators in cellular processes and the purpose of this study is to identify novel miRNA regulators implicated in human aortic VSMC proliferation and migration.

RESULTS

To identify miRNAs that are differentially expressed in human VSMCs, we performed miRNA microarray analysis in human aortic smooth muscle cells (SMCs) at different time points after platelet-derived growth factor (PDGF) stimulation. Here, we identified microRNA-638 (miR-638) as a transcript that was one of the most significantly down-regulated in human VSMCs after PDGF stimulation. Furthermore, we confirmed, by Quantitative RT-PCR, that miR-638 is highly expressed in human VSMCs, and its expression is markedly down-regulated in a dose- and time-dependent manner upon PDGF treatment. Consistent with a critical role in SMC proliferation, we found that miR-638 expression was significantly up-regulated in human VSMCs cultured in differentiation medium, a condition that inhibits SMC proliferation. Furthermore, we identified the orphan nuclear receptor NOR1 as a downstream target gene product of miR-638 and down-regulation of NOR1 is critical for miR-638-mediated inhibitory effects on PDGF-induced cyclin D1 expression, cell proliferation, and migration in human aortic SMCs.

CONCLUSIONS

These results indicate that miR-638 is a key molecule in regulating human VSMC proliferation and migration by targeting the NOR1/cyclin D pathway and suggest that specific modulation of miR-638 in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

High affinity binding of platelet-derived growth factor (PDGF) has been proposed to involve the interaction of the dimeric PDGF ligand with two receptor subunits, designated alpha and beta. We have cloned and expressed a human PDGF receptor cDNA which differs in sequence from the beta-subunit and which has the PDGF binding properties and monoclonal antibody recognition, predicted for the alpha-subunit. Scatchard analysis indicated that PDGF-AA and PDGF-AB bound to transfected alpha-subunits with affinities of Kd = 0.06 and 0.05 nM, respectively. PDGF-BB bound with a significantly lower affinity (Kd = 0.4 nM). Nevertheless, this affinity is still great enough to mediate substantial PDGF-BB binding at physiological concentrations and would be considered to be "high affinity." We have used wild-type and kinase-inactive human beta-subunits to show that PDGF binding promotes receptor subunit dimerization in intact cells. In addition, we found that PDGF stimulates tyrosine phosphorylation of the kinase-inactive beta-subunit when it is expressed with alpha-subunits. The kinase-inactive beta-subunits were phosphorylated at tyrosine 857 and 751, the major phosphorylation sites of the wild-type beta-subunit, indicating either that intra- and intermolecular phosphorylation occurs on the same sites, or that a significant fraction of receptor tyrosine phosphorylation is intermolecular.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase-derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB-induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase-derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK.

Directed cell migration is essential for a variety of important biological processes ranging from development and angiogenesis to metastasis. Ras plays a pivotal role in the signaling cascade that governs chemotaxis of fibroblasts toward platelet-derived growth factor-BB (PDGF-BB). Ras activates multiple downstream pathways, which include the extracellular signal-regulated kinase (ERK), Rac, and Ral signaling cascades. We therefore investigated the role of the Rac and ERK pathways in cell migration. We showed that migration of fibroblasts toward PDGF-BB is inhibited by expression of dominant negative Asn-17 Rac1. Blocking of the ERK pathway by either expression of dominant negative Ala-218/Ala-222-mitogen-activated protein kinase kinase (A218/A222-MEK1) or by a MEK-specific inhibitor did not inhibit migration toward PDGF-BB. In contrast, migration toward soluble fibronectin was suppressed by inhibition of the ERK pathway but not by Asn-17 Rac1 expression. These results indicate that directed cell migration mediated by different receptor classes in response to different ligands differentially utilizes the Rac and ERK pathways and suggest that Rac might play a critical role in pathological processes such as angiogenesis and metastasis.

The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE-statin for vascular endothelial-statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE-statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE-statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)-like modules. VE-statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE-statin inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE-statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

The human endometrium is a dynamic remodeling tissue undergoing more than 400 cycles of regeneration, differentiation and shedding during a woman's reproductive years. The co-ordinated and sequential actions of estrogen and progesterone direct these major remodeling events preparing a receptive endometrium for blastocyst implantation on a monthly basis. Adult stem/progenitor cells are likely responsible for endometrial regeneration. Functional approaches have been used to identify candidate endometrial stem/progenitor cells, as there are no specific stem cell markers. Rare populations of human endometrial epithelial and stromal colony-forming cells/units (CFU) and side population (SP) cells have been identified. Several growth factors are required for CFU activity: epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and platelet-derived growth factor BB (PDGF-BB) for both epithelial and stromal CFU, and basic fibroblast growth factor (bFGF) for stromal, but not epithelial CFU. A sub-population of human endometrial stromal cells with mesenchymal stem cell properties of CFU activity and multilineage (fat, muscle, cartilage and bone) differentiation have been isolated by their co-expression of CD146 and PDGF-receptor beta. Candidate epithelial and stromal stem/progenitor cells have been identified in mouse endometrium as rare label retaining cells (LRCs) in the luminal epithelium and as perivascular cells at the endometrial-myometrial junction, respectively. While epithelial and most stromal LRC do not express estrogen receptor alpha (Esr1), they rapidly proliferate on estrogen stimulation, most likely mediated by neighbouring Esr1-expressing niche cells. It is likely that these newly identified endometrial stem/progenitor cells may play key roles in the development of gynecological diseases associated with abnormal endometrial proliferation such as endometriosis and endometrial cancer.

The roles of polypeptide growth factors in promoting wound healing and in directing the specificity and sequence of responses of different tissues in wounds are little understood. We investigated the influence of four growth factors on the rates of healing of a novel full thickness dermal ulcer placed on an avascular base in the rabbit ear. The wound model precludes significant wound contraction and requires new granulation tissue and epithelial cells for healing to originate centripetally. 5 micrograms (7-31 pmol/mm2) of platelet-derived growth factor-B chain (PDGF-BB), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) applied locally at the time of wounding resulted in a twofold increase in complete reepithelialization of treated wounds (PDGF-BB, P = 0.02 chi square analysis; bFGF, P = 0.04; EGF, P = 0.05); transforming growth factor (TGF)-beta 1 significantly inhibited reepithelialization (P = 0.05). Both PDGF-BB and TGF-beta 1 uniquely increased the depth and area of new granulation tissue (P less than 0.005), the influx of fibroblasts, and the deposition of new matrix into wounds. Explants from 7-d old PDGF-BB-treated wounds remained metabolically far more active than controls, incorporating 473% more [3H]thymidine into DNA (P = 0.05) and significantly more [3H]leucine and [3H]proline into collagenase-sensitive protein (P = 0.04). The results establish that polypeptide growth factors have significant and selective positive influences on healing of full thickness ulcers in the rabbit.

A hallmark of smooth muscle cell (SMC) phenotypic switching is suppression of SMC marker gene expression. Although myocardin has been shown to be a key regulator of this process, the role of its related factors, MKL1 and MKL2, in SMC phenotypic switching remains unknown. The present studies were aimed at determining if: 1) MKL factors contribute to the expression of SMC marker genes in cultured SMCs; and 2) platelet-derived growth factor-BB (PDGF-BB)-induced repression of SMC marker genes is mediated by suppression of MKL factors. Results of gain- and loss-of-function experiments showed that MKL factors regulated the expression of single and multiple CArG [CC(AT-rich)(6)GG]-containing SMC marker genes, such as smooth muscle (SM) alpha-actin and telokin, but not CArG-independent SMC marker genes such as smoothelin-B. Treatment with PDGF-BB reduced the expression of CArG-containing SMC marker genes, as well as myocardin expression in cultured SMCs, while it had no effect on expression of MKL1 and MKL2. However, of interest, PDGF-BB induced the dissociation of MKL factors from the CArG-containing region of SMC marker genes, as determined by chromatin immunoprecipitation assays. This dissociation was caused by the competition between MKL factors and phosphorylated Elk-1 at early time points, but subsequently by the reduction in acetylated histone H4 levels at these promoter regions mediated by histone deacetylases, HDAC2, HDAC4, and HDAC5. Results provide novel evidence that PDGF-BB-induced repression of SMC marker genes is mediated through combinatorial mechanisms, including downregulation of myocardin expression and inhibition of the association of myocardin/MKL factors with CArG-containing SMC marker gene promoters.

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.

BACKGROUND

The goals of this study were to characterize urine-derived stem cells obtained from the upper urinary tract (uUSC), induce these cells to differentiate into urothelial and smooth muscle cells, and determine whether they could serve as a potential stem cell source for bladder tissue engineering.

METHODS

Urine samples were collected from five patients with normal upper urinary tracts during renal pyeloplasty. Cells were isolated from this urine and extensively expanded in vitro.

RESULTS

The mean population doubling of uUSC was 46.5±7.7. The uUSC expressed surface markers associated with mesenchymal stem cells and pericytes. These cells could differentiate into smooth muscle-like cells that expressed smooth muscle-specific gene transcripts and proteins, including α-smooth muscle actin, desmin, and myosin, when exposed to TGF-β1 and PDGF-BB. In a collagen lattice assay, these myogenic-differentiated uUSC displayed contractile function that was similar to that seen in native smooth muscle cells. Urothelial-differentiated uUSC expressed urothelial-specific genes and proteins such as uroplakin-Ia and -III, cytokeratin (CK)-7, and CK-13.

CONCLUSIONS

uUSC possess expansion and differentiation (urothelial and myogenic) capabilities, and can potentially be used as an alternative cell source in bladder tissue engineering for patients needing cystoplasty.

We recently reported that the first detectable expression of SMC-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells was restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). The objectives of this study were to examine more closely the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and to specifically test whether rhoA-GTPase signaling is required for CoSMC differentiation. We report here that PDGF-BB stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22alpha, and SMgamma(actin) (SMgammaA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocked PDGF-BB-induced EMT, prevented actin reorganization into stress fibers, and inhibited CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocked EMT, prevented the appearance of calponin and SMgammaA-positive cells, and abolished expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI or vehicle and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI failed to form CoSMCs in vivo. Time course studies traced this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA-RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells.

Modulation of cytokine expression represents a potentially useful approach for the treatment of idiopathic pulmonary fibrosis (IPF). To identify potential targets for such intervention, semi-quantitative reverse transcriptase-polymerase chain reaction was used to compare the expression of messenger ribonucleic acids (mRNAs) coding for 17 cytokines in lung tissue obtained from patients with IPF at the time of diagnosis and control subjects. Some cytokines were also studied at the protein level by immunohistochemical techniques. mRNAs coding for all of the cytokines evaluated were detected in both control and fibrotic lung samples. Only transforming growth factor (TGF)-beta and interleukin (IL)-10 mRNAs were quantitatively increased in lung biopsies from patients with IPF compared with those of controls, results confirmed at the protein level by immunohistochemistry. Although mRNAs for platelet-derived growth factor (PDGF)-BB and keratinocyte growth factor (KGF) were expressed in similar amounts in lungs from patients with IPF and controls, localised accumulation of both factors was also observed in IPF. Hyperplastic alveolar epithelial cells were a prominent source of cytokines, where IL-10, PDGF-BB and KGF were present in increased amounts, although increased accumulation in fibroblasts, smooth-muscle cells and matrix components was also observed (PDGF-BB, TGF-beta). These results offer new insights into the cytokines produced in the lung in idiopathic pulmonary fibrosis and suggest that modulation of the production of transforming growth factor-beta and interleukin-10 may represent a potentially useful therapeutic strategy for this disabling disease.

Platelet-derived growth factor-BB (PDGF-BB) is a well-characterized growth factor displaying potent biological effects on angiogenesis. Recent studies reveal that overexpression of PDGF-BB within tumors results in increased pericyte coverage, suggesting that PDGF-BB signaling is also essential for the cancerous pericyte recruitment process. However, the molecular mechanism underlying this regulation remains obscure. In the current study, we show that tumor-derived PDGF-BB induces SDF-1alpha expression in endothelial cells (EC), resulting in the formation of SDF-1alpha chemotaxis gradient, which coincides with the PDGF-BB-induced pericyte recruitment during angiogenesis. PDGF-BB dramatically up-regulates SDF-1alpha secretion through the activation of PDGFRbeta in tumor-associated ECs, whereas this up-regulation can be substantially inhibited by either blockade of the phosphatidylinositol 3-kinase/Akt/mTOR pathway with chemical inhibitors or the inactivation of HIF-1alpha through small interfering RNA interference. On the other hand, we reveal that SDF-1alpha can increase pericytes motility in vitro. Blockade of the SDF-1alpha/CXCR4 axis prevents the PDGF-BB-induced pericyte recruitment not only in three in vitro recruitment models but also in the PDGF-BB-overexpressing tumor xenograft models. These results highlight that the involvement of SDF-1alpha/CXCR4 axis is essential for the pericyte recruitment within the PDGF-BB-overexpressing tumors and raise the possibility that blockade of the SDF-1alpha/CXCR4 axis may provide a therapeutic synergy with antiangiogenic molecules that selectively target ECs.