Transforming Growth Factor-β1 Induces Transdifferentiation of Myoblasts into Myofibroblasts via Up-Regulation of Sphingosine Kinase-1/S1P3 Axis

The pleiotropic cytokine transforming growth factor (TGF)-β1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFβ1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFβ1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFβ1. Moreover, inhibition of S1P₃, which became the highest expressed S1PR after TGFβ1 challenge, strongly attenuated the profibrotic response to TGFβ1. Furthermore, downstream of S1P₃, Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFβ1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P₂, consequently to TGFβ1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFβ1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.     

Bradykinin mediates myogenic differentiation in murine myoblasts through the involvement of SK1/Spns2/S1P2 axis

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P₂ receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.     

Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.     

FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle,breast cancer and androgen-independent prostate cancer cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod?) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P_2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P_2/3 receptors.     

Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P2 receptor axis

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P₂ receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.     

Cannabinoid CB1 receptor inhibition decreases vascular smooth muscle migration and proliferation

Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli and chemoattractants such as platelet-derived growth factor (PDGF) are key events in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation and migration in various cell types through cannabinoid receptors. Here we investigated the effects of CB₁ receptor antagonist rimonabant (SR141716A), which has recently been shown to have anti-atherosclerotic effects both in mice and humans, on PDGF-induced proliferation, migration, and signal transduction of human coronary artery smooth muscle cells (HCASMCs). PDGF induced Ras and ERK 1/2 activation, while increasing proliferation and migration of HCASMCs, which were dose dependently attenuated by CB₁ antagonist, rimonabant. These findings suggest that in addition to improving plasma lipid alterations and decreasing inflammatory cell migration and inflammatory response, CB₁ antagonists may exert beneficial effects in atherosclerosis and restenosis by decreasing vascular smooth muscle proliferation and migration.     

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.     

Disruption of integrin α5β1 signaling does not impair PDGF-BB-mediated stimulation of the extracellular signal-regulated kinase pathway in smooth muscle cells

Smooth muscle cell (SMC) proliferation is dependent on both anchorage to the extracellular matrix by integrins and the presence of growth factors. Integrins and growth factor receptors transduce signals that seem to converge on the extracellular signal-regulated (ERK) pathway, but the molecular basis for this interaction is not known. SMC proliferation has previously been shown to be supported by culture on fibronectin (FN), whereas cells cultured on laminin (LN) are growth inhibited. In the present study, we examined the mitogenic response to platelet-derived growth factor BB (PDGF-BB) in baboon SMCs cultured on FN vs. LN. Induction of DNA synthesis and the activity of ERK and the ERK activating kinase MKK-1 were reduced only slightly after stimulation with PDGF-BB in cells cultured on LN vs. those cultured on FN. We tested the possibility that endogenous FN secretion contributes to the ability of the cells to respond to PDGF stimulation during culture on LN. Inhibition of interactions between FN and integrin α₅β₁ by the competitive GRGDSP-peptide or anti-α₅ integrin antibody restricted cell spreading, reduced cell-surface staining for α₅β₁ and FN fibrils, and inhibited PDGF-BB-induced DNA synthesis. These results showed that SMC growth on LN required a provisional FN matrix. Although disruption of interactions between α₅β₁ and FN by the GRGDSP-peptide prevented PDGF-BB-induced DNA synthesis, neither ERK activity nor translocation of ERKs into the nucleus was inhibited. These results show that integrins regulate SMC growth through pathways that function in parallel with, but distinct from, growth factor-mediated ERK signaling. J. Cell. Physiol. 172:109–116, 1997. © 1997 Wiley-Liss, Inc.     

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

Voltage-dependent K⁺ channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G₁-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.     

Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGF beta receptor-sphingosine 1-phosphate receptor complexes in airway smooth muscle cells

Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF beta receptor-S1P(1) receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P(1) antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF beta receptor-S1P(1) receptor complex. Third, treatment of cells with antisense S1P(1) receptor plasmid construct reduced the PDGF- and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF beta receptors and S1P(1) receptors, which was required for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF beta receptor-S1P(1) receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-protein-coupled receptor agonists and/or sequential release and action of S1P in response to PDGF.     

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT₁ receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT₁ receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT₁ receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar¹-Ile⁴-Ile⁸]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT₁ receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT₁ receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.     

Growth factor-induced proliferation of human fibroblasts in serum-free culture depends on cell density and extracellular calcium concentration

Human neonatal skin fibroblasts plated sparsely in MCDB 105 traversed a complete cell cycle in the absense of serum or serum-derived proteins. Addition of pure PDGF did not significantly increase entrance into S phase as revealed by ³H-thymidine labeling index or clonal growth on palladium islands. In subphysiologic Ca²⁺ concentrations or in the presence of a calmodulin inhibitor, W7, proliferation in the absence of growth factors ceased and PDGF became mitogenic. In contrast, confluent fibroblast cultures were stimulated by PDGF in physiologic Ca²⁺ concentrations. This was also the case with sparse adult skin fibroblast cultures while a fetal strain entered S in the absence of PDGF even in low extracellular Ca²⁺ concentrations. EGF gave similar results as PDGF in all experiments performed. This proposes a similar role for the two growth factors in the cell cycle. However, a difference in the mechanisms of action of PDGF and EGF is indicated by the fact that PDGF and EGF were additive at optimal concentrations when maximal growth response by a single growth factor was restricted by a subphysiologic extracellular Ca²⁺ concentration.     

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.     

Sphingosine 1-phosphate signalling and termination at lipid phosphate receptors

Sphingosine 1-phosphate (S1P) is a polar lysophospholipid metabolite that is stored in platelets and released upon their activation. However, diverse stimuli such as growth factors, cytokines, G-protein coupled receptor (GPCR) agonists and antigens have been shown to increase sphingosine kinase activity and S1P formation in other cell types, such as smooth muscle. Indeed, S1P has been implicated in the regulation of several important cellular processes, such as proliferation, differentiation, apoptosis and migration in these cells. Over the past few years, there has been a major advance in our understanding of how S1P can act as an intercellular mediator by binding to a new class of G-protein coupled receptors to regulate cell function. This review focuses on the enzymatic regulation of S1P formation and degradation and its interaction with a novel tethered receptor complex containing the S1P receptor (S1P(1)) and the platelet-derived growth factor (PDGF) beta receptor. This tethered receptor complex enables coincident integrative signalling to p42/p44 MAPK. This is compared with a sequential model in which PDGF promotes S1P release, which in turn acts on S1P(1) to promote Rac signalling.     

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: Possible role in regulating cell division

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P₅, a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, G_i and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P₅ (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P₅ may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P₅ to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P₅ receptor might function as an intracellular target of S1P linked to regulation of mitosis.     

Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells.

We have studied the effect of transforming growth factor beta 1 (TGF-beta 1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF-beta 1 treatment of vascular SMC induced a prolonged increase in steady-state mRNA levels of thrombospondin as well as alpha 1 (IV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet-derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF-beta 1 was able to directly enhance expression of thrombospondin as well as the growth-related genes c-fos and c-myc, and induced c-fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF-beta 1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF-beta 1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c-myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF-beta 1 alone was observed. Our results strongly suggest that induction of thrombospondin expression by TGF-beta 1 and by PDGF occurs by distinct mechanisms. In addition, that TGF-beta 1 can enhance PDGF-induced mitogenesis may be due to the ability of TGF-beta 1 to directly induce the expression of thrombospondin, c-fos, c-myc, and the PDGF beta-receptor.     

Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Extracellular matrix is a source of mitogenically active platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A_s (short splice version) is secreted, PDGF A_L (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and η-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. © 1996 Wiley-Liss, Inc.     

Endothelin and PDGF do not stimulate peripheral blood monocyte chemotaxis, adhesion to endothelium, and superoxide production

Endothelin is a potent vasoconstrictory endothelial-derived peptide which can induce smooth muscle proliferation and therefore may be proatherogenic. Platelet-derived growth factor is also a potent mitogenic vasoconstrictory protein which is proatherogenic. We report that neither endothelin nor PDGF stimulate superoxide production, or monocyte adhesion to porcine aortic endothelial cell monolayers; additionally endothelin is not a chemoattractant factor for monocytes. If endothelin and PDGF are important in atherogenesis it is unlikely that a monocyte response to these mediators is involved.