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At least 343 records · Page 19Linked to original sources
PubMed2006

Platelet-derived growth factor-BB transactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells.

Platelet-derived growth factor (PDGF) is a major stimulant for smooth muscle cell migration and proliferation, and blockade of PDGF receptors in vivo reduces intimal growth after arterial injury. Signalling by PDGF receptors has been extensively studied and involves multiple signal transduction pathways. These include ras/Extracellular Signal Regulated Kinase (ERK), a pathway critical for controlling cell cycle progression. We have recently discovered that release of fibroblast growth factor 2 and the subsequent activation of fibroblast growth factor receptor 1 are required for maximal induction by PDGF of ERK and of human smooth muscle cell proliferation. This review summarizes our latest findings and discusses the potential implications of this novel signaling mechanism for restenosis.

Becaplermin↗
PubMed2006

Resting smooth muscle cells as a model for studying vascular cell activation.

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.

Animals↗
PubMed2006

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this second article, we investigate the platelet-associated features of this biomaterial. During PRF processing by centrifugation, platelets are activated and their massive degranulation implies a very significant cytokine release. Concentrated platelet-rich plasma platelet cytokines have already been quantified in many technologic configurations. To carry out a comparative study, we therefore undertook to quantify PDGF-BB, TGFbeta-1, and IGF-I within PPP (platelet-poor plasma) supernatant and PRF clot exudate serum. These initial analyses revealed that slow fibrin polymerization during PRF processing leads to the intrinsic incorporation of platelet cytokines and glycanic chains in the fibrin meshes. This result would imply that PRF, unlike the other platelet concentrates, would be able to progressively release cytokines during fibrin matrix remodeling; such a mechanism might explain the clinically observed healing properties of PRF.

Adult↗
PubMed2006

Antiproliferative activity of NQ304, a synthetic 1,4-naphthoquinone, is mediated via the suppressions of the PI3K/Akt and ERK1/2 signaling pathways in PDGF-BB-stimulated vascular smooth muscle cells.

Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of atherosclerosis. The aim of this study was to assess the effect of NQ304 [2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone], a newly synthesized 1,4-naphthoquinone derivative, on the proliferation of PDGF-BB-stimulated rat aortic VSMCs. Antiproliferative effects of NQ304 on rat aortic VSMCs were examined by direct cell counting and by using [(3)H] thymidine incorporation assays. It was found that NQ304 potently the growth of VSMCs. Preincubation with NQ304 (1-10 microM) significantly inhibited proliferation and DNA synthesis of 50 ng/ml PDGF-BB-stimulated rat aortic VSMCs in a concentration-dependent manner. In addition, we investigated the mechanism of proliferation suppression by NQ304 in PDGF-BB-stimulated rat aortic VSMCs, and found that PDGF-BB-stimulated immediate-early gene expression (c-fos), activator protein (AP)-1 activation, extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and Akt kinase were significantly inhibited by NQ304. An examination of the suppressive effects of NQ304 on PDGF-BB-stimulated VSMC cycle progression showed that NQ304 (10 microM) induced the G1 phase arrest of PDGF-BB-stimulated cell cycle progression by elevating p21(cip1) mRNA expression. These findings suggest that the inhibitory effects of NQ304 on DNA synthesis, proliferation, and cell cycle progression on PDGF-BB-stimulated VSMCs are mediated via the downregulations of AP-1 activation and c-fos expression achieved in turn via the suppressions of the phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2 signaling pathways.

Animals↗
PubMed2004

The mesenchymal alpha11beta1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens.

alpha11beta1 constitutes the most recent addition to the integrin family and has been shown to display a binding preference for interstitial collagens found in mesenchymal tissues. We have previously observed that when alpha11beta1 integrin is expressed in cells lacking endogenous collagen receptors, it can mediate PDGF-BB-dependent chemotaxis on collagen I in vitro. To determine in which cells PDGF and alpha11beta1 might cooperate in regulating cell migration in vivo, we studied in detail the expression and distribution of alpha11 integrin chain in mouse embryos and tested the ability of PDGF isoforms to stimulate the alpha11beta1-mediated cell migration of embryonic fibroblasts. Full-length mouse alpha11 cDNA was sequenced and antibodies were raised to deduced alpha11 integrin amino acid sequence. In the embryonic mouse head, alpha11 protein and RNA were localized to ectomesenchymally derived cells. In the periodontal ligament, alpha11beta1 was expressed as the only detectable collagen-binding integrin, and alpha11beta1 is thus a major receptor for cell migration and matrix organization in this cell population. In the remainder of the embryo, the alpha11 chain was expressed in a subset of mesenchymal cells including tendon/ligament fibroblasts, perichondrial cells, and intestinal villi fibroblasts. Most of the alpha11-expressing cells also expressed the alpha2 integrin chain, but no detectable overlap was found with the alpha1 integrin chain. In cells expressing multiple collagen receptors, these might function to promote a more stable cell adhesion and render the cells more resistant to chemotactic stimuli. Wild-type embryonic fibroblasts activated mainly the PDGF beta receptor in response to PDGF-BB and migrated on collagens I, II, III, IV, V, and XI in response to PDGF-BB in vitro, whereas mutant fibroblasts that lacked alpha11beta1 in their collagen receptor repertoire showed a stronger chemotactic response on collagens when stimulated with PDGF-BB. In the cellular context of embryonic fibroblasts, alpha11beta1 is thus anti-migratory. We speculate that the PDGF BB-dependent cell migration of mesenchymal cells is tightly regulated by the collagen receptor repertoire, and disturbances of this repertoire might lead to unregulated cell migration that could affect normal embryonic development and tissue structure.

Amino Acid Sequence↗
PubMed2003

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1.

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.

Animals↗
PubMed2004

Wrch1 is a GTPase-deficient Cdc42-like protein with unusual binding characteristics and cellular effects.

The Rho family of small GTPases controls many biological processes, including cytoskeletal regulation, membrane trafficking, cell adhesion, cell polarization, transcriptional activity, apoptosis, and cell proliferation. Wrch1, which belongs to the Cdc42 subfamily, is one of the least characterized family member. Despite its homology to other Cdc42-like proteins, we found that Wrch1 has unique characteristics. Biochemical experiments showed that Wrch1 has no detectable GTPase activity in vitro and that its intrinsic nucleotide exchange rate is very high in comparison to Cdc42. Furthermore, NIH3T3 cells transiently transfected with Wrch1 showed an up-rounded, retracted phenotype. In addition, Wrch1 was shown to be more efficient than Cdc42 in triggering the formation of filopodia. Serum stimulation of cells expressing Wrch1 induces vigorous membrane blebbing, a phenomenon dependent on the activity of ROCK. In a search for proteins interacting with Wrch1, PAK1 and NCKbeta were identified as binding partners. Interestingly, the interaction to NCKbeta was shown to be mediated via PxxP motifs present in an N-terminal extension of Wrch1 to the second and third SH3 domains of NCKbeta.

3T3 Cells↗
PubMed2004

Differential activation of vascular genes by hypoxia in primary endothelial cells.

Changes in the local environment, such as reduced oxygen tension (hypoxia), elicit transcriptional activation of a variety of genes in mammalian cells. Here we have analyzed the effect of hypoxia in different vascular endothelial cells (ECs) with emphasis on hypoxia-regulated transcription factors and genes of importance for blood vessel dynamics. While hypoxia induced the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) in all endothelial cells tested, the closely related HIF-2alpha protein was markedly induced in microvascular/capillary endothelial cells, but only weakly or not at all in artery and vein endothelial cells. Furthermore, microvascular/capillary endothelial cells responded to hypoxia with increased number of transcripts encoding vascular endothelial growth factor-A (VEGF-A), VEGF receptor-2, the angiopoietin receptor Tie2, platelet-derived growth factor-B (PDGF-B), and inducible nitric oxide synthase (iNOS). In vein endothelial cells, hypoxia instead increased transcripts encoding lymphatic vascular components VEGF-C, -D, and VEGF receptor-3. Finally, reduced VEGF receptor levels and phosphorylation indicated establishment of a functional autocrine VEGF-A loop in hypoxic endothelial cells. Our results show that endothelial cells, derived from different vascular beds, mount different transcriptional responses to changes in oxygen tension.

Animals↗
PubMed2004

Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines.

OBJECTIVES: We determined the expression of platelet-derived growth factor (PDGF), PDGF-receptor (PDGF-R), and phosphorylated PDGF-R (p-PDGF-R) on tumor cells and tumor-associated endothelial cells in clinical specimens of human ovarian carcinoma and human ovarian cancer cells growing in culture and in the peritoneal cavity of nude mice. METHODS: Ten specimens of high-grade serous ovarian carcinoma were analyzed using immunohistochemistry (IHC). IHC was used to detect ligand and receptor expression in the human ovarian cancer cells from Hey A8 and SKOV3ip1 growing in culture. Cells from these lines were also implanted orthotopically into the peritoneal cavity of nude mice. IHC was used to determine ligand and receptor expression in tumors that formed in the peritoneal cavity. RESULTS: All 10 evaluable samples expressed both PDGF AA and BB on tumor cells. Tumor cells were positive for PDGF-Ralpha in 10/10 samples, PDGF-Rbeta in 8/10 samples, p-PDGF-Ralpha in 6/10 samples, and p-PDGF-Rbeta in 4/10 samples. p-PDGF-Ralpha was positive in 4/10 tumor-associated endothelial cell samples and p-PDGF-Rbeta was positive in 3/10 samples. Human ovarian cancer cells expressed PDGF, PDGF-R, and p-PDGF-R when growing in culture or in the peritoneal cavity of nude mice. PDGF-R and p-PDGF-R were also present on tumor-associated endothelial cells as demonstrated by simultaneous staining with CD31 antibody. CONCLUSIONS: PDGF and the corresponding receptors were expressed in autochthonous human ovarian cancer lesions on both tumor cells and tumor-associated endothelial cells. The ligand and receptor were also present on Hey A8 and SKOV3ip1 human ovarian cancer cells growing in vitro and in the peritoneal cavity of nude mice.

Animals↗
PubMed2006

PDGF-BB enhances monocyte chemoattractant protein-1 mRNA stability in smooth muscle cells by downregulating ribonuclease activity.

Platelet-derived growth factor (PDGF) has protean manifestations, including the regulation of growth and migration, in many cell types. We have previously reported that PDGF-BB induces the accumulation of monocyte chemoattractant protein (MCP)-1 mRNA in smooth muscle cells (SMC), in large part due to an increase in mRNA stability. To elucidate the mechanism by which PDGF-BB stabilizes MCP-1 mRNA, we have employed in vitro RNA gel mobility shift and decay assays. Cytoplasmic extracts from PDGF-BB-treated SMC increased the half-life of in vitro transcribed MCP-1 mRNA from approximately 45 min to >2 h. PDGF-BB-inhibitable degradation was not dependent on specific regions of the MCP-1 mRNA and was equally effective on a variety of in vitro transcribed mRNAs. Angiotensin II had a similar effect on MCP-1 mRNA stability, whereas tumor necrosis factor-alpha and basic fibroblast growth factor did not. The PDGF-BB-inhibitable RNAse activity was active at pH 6.6 and heat stable, but was sensitive to proteinase K. Extracts from PDGF-BB- or angiotensin II-treated cells inhibited the RNAse activity of control extracts, suggesting that the effect of PDGF-BB and angiotensin II are due to activation of a soluble inhibitor of the RNAse. The effect of PDGF-BB was blocked by inhibitors of tyrosine phosphorylation, but not by inhibitors of phosphatidylinositol 3-kinase or mitogen-activated protein kinases. These studies provide new insights into the mechanisms by which PDGF-BB enhances mRNA accumulation.

Angiotensin II↗
PubMed2001

Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice.

Receptor for advanced glycation end-products (RAGE), and two of its ligands, AGE and EN-RAGEs (members of the S100/calgranulin family of pro-inflammatory cytokines), display enhanced expression in slowly resolving full-thickness excisional wounds developed in genetically diabetic db+/db+ mice. We tested the concept that blockade of RAGE, using soluble(s) RAGE, the extracellular ligand-binding domain of the receptor, would enhance wound closure in these animals. Administration of sRAGE accelerated the development of appropriately limited inflammatory cell infiltration and activation in wound foci. In parallel with accelerated wound closure at later times, blockade of RAGE suppressed levels of cytokines; tumor necrosis factor-alpha; interleukin-6; and matrix metalloproteinases-2, -3, and -9. In addition, generation of thick, well-vascularized granulation tissue was enhanced, in parallel with increased levels of platelet-derived growth factor-B and vascular endothelial growth factor. These findings identify a central role for RAGE in disordered wound healing associated with diabetes, and suggest that blockade of this receptor might represent a targeted strategy to restore effective wound repair in this disorder.

Animals↗
PubMed2004

Thy-1 expression regulates the ability of rat lung fibroblasts to activate transforming growth factor-beta in response to fibrogenic stimuli.

Distinct subpopulations of fibroblasts contribute to lung fibrosis, although the mechanisms underlying fibrogenesis in these subpopulations are not clear. Differential expression of the glycophosphatidylinositol-linked protein Thy-1 affects proliferation and myofibroblast differentiation. Lung fibroblast populations selected on the basis of Thy-1 expression by cell sorting were examined for responses to fibrogenic stimuli. Thy-1 (-) and Thy-1 (+) fibroblast populations were treated with platelet-derived growth factor-BB, interleukin-1beta, interleukin-4, or bleomycin and assessed for activation of transforming growth factor (TGF)-beta, Smad3 phosphorylation, and alpha-smooth muscle actin and fibronectin expression. Thy-1 (-) fibroblasts responded to these stimuli with increased TGF-beta activity, Smad3 phosphorylation, and expression of alpha-smooth muscle actin and fibronectin, whereas Thy-1 (+) fibroblasts resisted stimulation. The unresponsiveness of Thy-1 (+) cells is not because of defective TGF-beta signaling because both subsets respond to exogenous active TGF-beta. Rather, Thy-1 (-) fibroblasts activate latent TGF-beta in response to fibrogenic stimuli, whereas Thy-1 (+) cells fail to do so. Defective activation is common to multiple mechanisms of TGF-beta activation, including thrombospondin 1, matrix metalloproteinase, or plasmin. Thy-1 (-) lung fibroblasts transfected with Thy-1 also become resistant to fibrogenic stimulation, indicating that Thy-1 is a critical biological response modifier that protects against fibrotic progression by controlling TGF-beta activation. These studies provide a molecular basis for understanding the differential roles of fibroblast subpopulations in fibrotic lung disease through control of latent TGF-beta activation.

Actins↗
PubMed2003

Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Adult↗
PubMed2003

Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells.

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.

Animals↗
PubMed2003

Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment.

Platelet-derived growth factor (PDGF)-B and its receptor (PDGF-R) beta are overexpressed in human gliomas and responsible for recruiting peri-endothelial cells to vessels. To establish the role of PDGF-B in glioma angiogenesis, we overexpressed PDGF-B in U87MG glioma cells. Although PDGF-B stimulated tyrosine phosphorylation of PDGF-Rbeta in U87MG cells, treatment with recombinant PDGF-B or overexpression of PDGF-B in U87MG cells had no effect on their proliferation. However, an increase of secreted PDGF-B in conditioned media of U87MG/PDGF-B cells promoted migration of endothelial cells expressing PDGF-R beta, whereas conditioned media from U87MG cells did not increase the cell migration. In mice, overexpression of PDGF-B in U87MG cells enhanced intracranial glioma formation by stimulating vascular endothelial growth factor (VEGF) expression in neovessels and by attracting vessel-associated pericytes. When PDGF-B and VEGF were overexpressed simultaneously by U87MG tumors, there was a marked increase of capillary-associated pericytes as seen in U87MG/VEGF(165)/PDGF-B gliomas. As a result of pericyte recruitment, vessels induced by VEGF in tumor vicinity migrated into the central regions of these tumors. These data suggest that PDGF-B is a paracrine factor in U87MG gliomas, and that PDGF-B enhances glioma angiogenesis, at least in part, by stimulating VEGF expression in tumor endothelia and by recruiting pericytes to neovessels.

Animals↗
PubMed2002

Blockade of platelet-derived growth factor or its receptors transiently delays but does not prevent fibrous cap formation in ApoE null mice.

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

Animals↗
PubMed2000

alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Actins↗
PubMed1999

Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers.

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.

Animals↗