Presence Of Platelet-derived Growth factor-BB Research Articles

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB

During corneal wound healing, keratocytes present within the corneal stroma become activated into a repair phenotype upon the release of growth factors, such as transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). The process of injury and repair can lead to changes in the mechanical properties of the tissue, and previous work has shown that the TGF-β1-mediated myofibroblast differentiation of corneal keratocytes depends on substratum stiffness. It is still unclear, however, if changes in stiffness can modulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in stiffness influence the proliferation and motility of primary corneal keratocytes treated with PDGF-BB. In the presence of PDGF-BB, cells on stiffer substrata exhibited a more elongated morphology and had higher rates of proliferation than cells in a more compliant microenvironment. Using a freeze-injury to assay cell motility, however, we did not observe any stiffness-dependent differences in the migration of keratocytes treated with PDGF-BB. Taken together, these data highlight the importance of biophysical cues during corneal wound healing and suggest that keratocytes respond differently to changes in ECM stiffness in the presence of different growth factors.

The protective effect of HOXA5 on carotid atherosclerosis occurs by modulating the vascular smooth muscle cell phenotype

The phenotypic change of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic form is a key player in atherogenic processes. Homeobox A5 (HOXA5), a transcription factor of the homeobox gene family, has been shown to regulate cell differentiation and morphogenesis. The present study was designed to clarify the involvement of HOXA5 in VSMC phenotypic transition in carotid atherosclerosis (CAS). Activated VSMCs in vitro and ApoE−/- mice in vivo were employed to determine HOXA5's function. Results showed that both the mRNA and protein expression levels of HOXA5 were decreased in platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Overexpression of HOXA5 suppressed VSMC conversion from a contractile to a synthetic type in the presence of PDGF-BB, as evidenced by increased contractile markers (calponin, α-SMA and SM22α) along with decreased synthetic markers (vimentin, PCNA and thrombospondin). PDGF-BB-induced proliferation and migration of VSMCs were recovered by HOXA5. Knockdown of HOXA5 had the opposite effect on VSMCs. In vivo, a CAS model was established using ApoE−/- mice fed with a Western-type diet and placing a perivascular carotid collar. We observed a significant reduction in HOXA5 in the carotid arteries of CAS mice. Similar to the in vitro results, HOXA5 overexpression reduced neointimal hyperplasia and plaque formation and inhibited VSMC dedifferentiation and migration. Furthermore, PPARγ was also downregulated in vitro and in vivo, and its antagonist GW9662 reversed HOXA5-mediated inhibition of VSMC dedifferentiation and migration. In summary, we suggest that HOXA5 protects against CAS progression by inhibiting VSMC dedifferentiation through activation of PPARγ.

Tryptanthrin Regulates Vascular Smooth Muscle Cell Phenotypic Switching in Atherosclerosis by AMP-Activated Protein Kinase/Acetyl-CoA Carboxylase Signaling Pathway.

Atherosclerosis (AS) is one of the most severe cardiovascular diseases involved in the phenotypic switching of vascular smooth muscle cells (VSMCs). Tryptanthrin is a natural product with broad biological activities. However, the effect of tryptanthrin on atherosclerotic progression is unclear. The aim of this study was to determine the role of tryptanthrin in AS and explore the potential mechanism. In vitro, primary VSMCs were stimulated with platelet-derived growth factor-BB (PDGF) to induce cell dedifferentiation. Treatment with tryptanthrin (5 μM or 10 μM) suppressed the proliferation and recovered the contractility of VSMCs in the presence of PDGF. The contractile proteins (α-smooth muscle actin, calponin, and SM22α) were increased, and the synthetic protein vimentin was decreased by tryptanthrin in PDGF-induced VSMCs. ApoE-/- mice fed with high-fat diet were used as an in vivo model of AS. Similarly, gavage administration of tryptanthrin (50 mg/kg or 100 mg/kg) attenuated VSMC phenotypic changes from a contractile to a synthetic state in aortic tissues of AS mice. The serum lipid level, atherosclerotic plaque formation, and arterial intimal hyperplasia were attenuated by tryptanthrin. Furthermore, tryptanthrin increased the expression levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) both in vitro and in vivo. Administration of compound C, an AMPK inhibitor, reversed the inhibitory effect of tryptanthrin on VSMC dedifferentiation in vitro. Thus, we demonstrate that tryptanthrin protects against AS progression through the inhibition of VSMC switching from a contractile to a pathological synthetic phenotype by the activation of AMPK/ACC pathway. It provides novel insights into AS prevention and treatment.

Mechanisms of Platelet-Derived Growth Factor-BB in Restoring HIV Tat-Cocaine-Mediated Impairment of Neuronal Differentiation.

Diminished adult neurogenesis is known to play a key role in the pathogenesis of diverse neurodegenerative disorders such as HIV-associated neurological disorders (HAND). Cocaine, often abused by HIV-infected patients, has been suggested to worsen HIV-associated CNS disease. Mounting evidence also indicates that HIV infection can lead not only to neuronal dysfunction or loss, but can also negatively impact neurogenesis, resulting in generation of fewer adult neural progenitor cells (NPCs) in the dentate gyrus of the hippocampus, brain area critical for memory and learning. The crucial role of platelet-derived growth factor-BB (PDGF-BB) in providing tropic support for the neurons as well as in promoting NPC proliferation has been demonstrated by us previously. However, whether PDGF-BB regulates neuronal differentiation especially in the context of HAND and drug abuse remains poorly understood. In this study, we demonstrate that pretreatment of rat hippocampal NPCs with PDGF-BB restored neuronal differentiation that had been impaired by HIV Tat and cocaine. To further study the intracellular mechanism(s) involved in this process, we examined the role of transient receptor potential canonical (TRPC) channels in mediating neuronal differentiation in the presence of PDGF-BB. TRPC channels are Ca2+-permeable, nonselective cationic channels that elicit a variety of physiological functions. Parallel but distinct ERK, Akt signaling pathways with downstream activation of CREB were found to be critical for neuronal differentiation. Pharmacological blocking of TRPC channels resulted in suppression of PDGF-mediated differentiation and PDGF-BB-induced activation of ERK and Akt, culminating also to inhibition of PDGF-induced activation of CREB. Taken together, these findings underpin the role of TRPC channel as a novel target regulating cell differentiation mediated by PDGF-BB. This finding could have implications for development of therapeutic interventions aimed at restoration of Tat and cocaine-mediated impairment of neurogenesis in drug abusing HAND patients.

Sandwich-format electrochemiluminescence assay for PDGF-BB using quantum dots–dendrimer nanocomposites as probe

This work describes a novel electrochemiluminescence aptasensor for highly sensitive detection of platelet-derived growth factor BB (PDGF-BB) using aptamer functionalized CdS quantum dots–polyamidoamine as probe (CdS QDs–PAMAM–Apt). CdS QDs–PAMAM nanocomposites were synthesized by one-pot synthesis in methanol. The prepared nanocomposites were linked with the NH2–aptamer 2 (Apt2) of PDGF-BB to form the CdS QDs–PAMAM–Apt2 probe by glutaraldehyde as coupling reagent. For constructing the aptasensor, MWCNTs–chitosan composites and NH2–aptamer 1 (Apt1) with the same base sequence as Apt2 were immobilized on the electrode by the self-assembled method to recognize the target protein PDGF-BB. In the presence of PDGF-BB, the structure of sandwiched format was formed between the Apt1 and the CdS QDs–PAMAM–Apt2 probe, thereby resulting in a proportional increase of ECL emission. Thanks to the efficient and stable ECL emission of CdS QDs–PAMAM dendrimer and the advantage of MWCNTs for accelerating the electron transfer, the highly sensitive detection of PDGF-BB with a detection limit of 0.13pM was achieved. The linear range is from 0.5pM to 1nM. The present protocol was applied to the analysis of PDGF-BB in human serum samples. The recoveries of PDGF-BB in human serum samples are 87.2–113% and RSD values are less than 3.6%.

Photoinduced electron transfer (PET) based label-free aptasensor for platelet-derived growth factor-BB and its logic gate application

Platelet-derived growth factor-BB (PDGF-BB) is often overexpressed in human malignant tumors as an indicator for tumor angiogenesis. Here by the photoinduced electron transfer (PET) between DNA-Ag fluorescent nanoclusters (NCs) and G-quadruplex/hemin complexes, we present a sensitive label-free fluorescent sensor for PDGF-BB. In the presence of PDGF-BB, the specific conjugation with its aptamer induced the conformational change of the duplex-like DNA sequence, releasing the G-quadruplex sequence part. Then in the presence of hemin and K+, the horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) was formed. With the electron transfer between the DNA-Ag NCs to the hemin Fe (III) center of HRP-DNAzyme, the PET occurred with a decrease in the fluorescence intensity of the DNA-Ag NCs. The detection performance such as selectivity, linear dynamic range, sensitivity, and the quenching capability of HRP-DNAzyme were estimated. The detection range for PDGF-BB is from 5×10−13 to 1×10−8M and the detection limit is 1×10−13M. The experimental results confirmed that the novel fluorescent aptasensor possessed a good sensitivity and high selectivity for PDGF-BB. In addition, the developed probe is nontoxic, label-free only involving one-step hybridization without sophisticated fabrication process. Furthermore, based on this quenching mode occurred by PDGF-BB and hemin, using PDGF-BB and hemin as inputs and the fluorescence signal as an output, a logic gate has been fabricated.

Aptamer-based Liquid Crystal Biosensor for Detection of Platelet-derived Growth Factor BB

Abstract A novel liquid crystal (LC) biosensor was developed for the detection of platelet-derived growth factor BB (PDGF-BB) based on the orientation changes of liquid crystals. One glass slide of the LC cell was first modified with the APTES/DMOAP ((3-aminopropyl)trimethoxysilane/N,N-dimethyl-N-octadecyl(3-aminopropyl) trimethoxysilyl chloride) self-assembled monolayer (SAM) to trigger the homeotropic alignment of LC molecules and thereby produced a black background optical image under the crossed polarized light, and then the specific aptamer of PDGF-BB was immobilized on SAM through glutaraldehyde crosslinking to construct a LC sensing substrate. In the presence of PDGF-BB, the stable triple helix structure of PDGF-BB aptamer was formed by the specific binding event and thus led to the target PDGF-BB was captured to the sensing substrate, making a visible optical change which could be observed under the crossed polarized light via the huge steric effect of the PDGF-BB on disrupting the LC alignment. However, in the absence of PDGF-BB, the low packing density of the PDGF-BB aptamer had little effect on disrupting the ordered alignment to the LC and caused the black background optical image which could also be observed under the crossed polarized light. An obvious optical change was observed when the concentration of target PDGF-BB was 5 nM. The proposed LC biosensing method permits the label-free detection of PDGF-BB with good selectivity and high sensitivity, making them sufficiently simple and particularly useful for low-cost screening bioassay performed away from central laboratories.

Amplified amperometric aptasensor for selective detection of protein using catalase-functional DNA–PtNPs dendrimer as a synergetic signal amplification label

In this work, we present a new strategy to construct an electrochemical aptasensor for sensitive detection of platelet-derived growth factor BB (PDGF-BB) based on the synergetic amplification of a three-dimensional (3D) nanoscale catalase (CAT) enzyme-functional DNA–platinum nanoparticles (PtNPs) dendrimer through autonomous layer-by-layer assembly. Firstly, polyamidoaminedendrimer (PAMAM) with a hyper-branched and three-dimensional structure was served as nanocarriers to coimmobilize a large number of PDGF-BB binding aptamer (PBA II) and ssDNA 1 (S1) to form PBA II–PAMAM–S1 bioconjugate. In the presence of PDGF-BB, the bioconjugate was self-assembled on the electrode by sandwich assay. Following that, the carried S1 propagated a chain reaction of hybridization events between CAT–PtNPs–S1 and CAT–PtNPs–ssDNA 2 (S2) to form a 3D nanoscale CAT-functional PtNPs-DNA dendrimer, which successfully immobilized substantial CAT enzyme and PtNPs with superior catalysis activity. In this process, the formed negatively charged double-helix DNA could cause the intercalation of hexaammineruthenium(III) chloride (RuHex) into the groove via electrostatic interactions. Thus, numerous RuHex redox probes and CAT were decorated inside/outside of the dendrimer. In the presence of H2O2 in electrolytic cell, the synergistic reaction of CAT and PtNPs towards electrocatalysis could further amplify electrochemical signal. Under optimal condition, the CAT–PtNPs–DNA dendrimer-based sensing system presented a linear dependence between the reduction peak currents and logarithm of PDGF-BB concentrations in the range of 0.00005–35nM with a relatively low detection limit of 0.02pM.

Aptamer-based portable biosensor for platelet-derived growth factor-BB (PDGF-BB) with personal glucose meter readout

A novel portable biosensor for sensitive and selective detection of platelet-derived growth factor-BB (PDGF-BB) had been developed based on the specific recognition between aptamer and protein using a personal glucose meter (PGM) as readout. In the presence of PDGF-BB, the primary aptamer of PDGF-BB which is bound to the surface of streptavidin magnespheres paramagnetic particles (SA-PMPs) reacts quantitatively with invertase-functionalized secondary aptamer of PDGF-BB to form a stable complex, resulting in the attachment of invertase on the SA-PMPs. Subsequently, the invertase catalyzes the hydrolysis of sucrose to produce a large amount of glucose and quantitative readout by the PGM. The enhanced signal of the PGM has a relationship with the concentration of PDGF-BB in the range of 1.0×10–14M~3.16×10−12M, and the detection limit is 2.9fM. The proposed portable biosensor had been successfully applied to assay the PDGF-BB in saliva samples.

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

ObjectiveCatecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. MethodsFibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. ResultsGingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. ConclusionsThese results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

A highly sensitive and selective aptasensor based on graphene oxide fluorescence resonance energy transfer for the rapid determination of oncoprotein PDGF-BB

Oncoprotein platelet derived growth factor-BB (PDGF-BB) is one of the most critical growth factors that regulates tumor growth and division. In this work, a highly sensitive and selective fluorescence resonance energy transfer (FRET) aptasensor for PDGF-BB detection based on the assembly of dye-labeled aptamer and graphene oxide (GO) is developed for the first time. Due to the non-covalent assembly between aptamer and GO, fluorescence quenching of the dye takes place because of FRET. In the presence of PDGF-BB, the binding between aptamer and PDGF-BB will disturb the interaction between aptamer and GO, and release the dye-labeled aptamer from the GO surface, resulting in restoration of the fluorophore fluorescence. Because of the high fluorescence quenching efficiency, unique structure, and electronic properties of GO, the GO aptasensor exhibits extraordinarily high sensitivity. We also demonstrate that two highly related molecular variants of PDGF (AA, AB) can be distinguished from PDGF-BB, which indicates the aptasensor has excellent selectivity. Such an aptasensor opens a rapid, selective and sensitive route for the detection of PDGF-BB and provides a promising strategy for other cancer-related proteins detections.

Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg-Gly-Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10-100 nM. FN-null cells cultured on recombinant CCBD (FNIII(8-11)) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII(8-11) contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII(8-11) and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration.

Negative and Positive Regulation of MAPK Phosphatase 3 Controls Platelet-derived Growth Factor-induced Erk Activation

MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.

Canine retinal angioblasts are multipotent

The purpose of this study was to culture and characterize endothelial cells and angioblasts, vascular precursors, from adult and neonatal dog retina and determine if angioblasts are committed to endothelial cell lineage or have the potential to be multipotent, i.e. express phenotypic characteristics of other vascular cell types. Endothelial cells were established from adult dog retina (ADREC) by the technique of Gitlin and D'Amore. For angioblasts, pieces of neonatal day 2 (P2) avascular peripheral retina were placed under coverslips until sufficient cells had explanted. All cells were maintained initially on hyaluronic acid (HA)/fibronectin (FN) substratum. Neonatal canine retinal angioblasts (NCRA) were maintained initially on retinal-derived growth factor with alpha-amino adipic acid to inhibit growth of Muller cells. Cell lines were characterized by enzyme histochemistry [menadione-dependent alpha glycerophosphate dehydrogenase (αGPDH), marker for angioblasts] and immunocytochemistry. Once characterized, cells were grown on FN, or collagens I or IV substrata and fed platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF-2). The phenotypic expression of a marker for endothelial cells [acetylated LDL (acLDL) uptake] or a marker for pericytes and smooth muscle cells, production of alpha smooth muscle actin (αSMA), was evaluated under those conditions. The canine retinal cell lines that were established had the following characteristics when maintained on serum and a retinal extract. Angioblasts had low expression of vWf and VEGF-R2 (two markers for canine endothelial cells), and very low uptake of acLDL but high expression of αGPDH and adenosine A2a receptors (A2aR) (two markers for canine angioblasts in vivo). ADREC had high expression of endothelial cell markers (vWf, VEGF-R2, and acLDL uptake) but minimal expression of αGPDH and A2aR. Both angioblasts and endothelial cells expressed CXCR4, a marker for hemangioblasts. Angioblasts grown on any of the substrata in the presence of FGF-2 had high uptake of acLDL and low expression of αSMA, while those grown in the presence of PDGF-BB had high expression of αSMA and low uptake of acLDL. In conclusion, angioblasts cultured from peripheral vascular retina have low expression of endothelial cell markers and high αGPDH and A2aR, markers for canine angioblasts in vivo. Angioblasts will internalize acLDL when maintained on FGF-2 and express αSMA when maintained on PDGF-BB, suggesting that they have the potential to become endothelial cells or pericytes, i.e. are multipotent.

Effect of serum withdrawal on the contribution of L‐type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.

Potent Inhibitory Effect of Sirolimus on Circulating Vascular Progenitor Cells

Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation. Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)-like cells outgrew from the culture of MNCs (1x10(6)) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell-like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9+/-5.8% of control, P<0.001). Sirolimus also exerted an inhibitory effect on endothelial cell-like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0+/-0.3 versus 1.0+/-0.2, P<0.05) with a decreased number of bone marrow-derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus. The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.

The mosaic receptor sorLA/LR11 binds components of the plasminogen-activating system and platelet-derived growth factor-BB similarly to LRP1 (low-density lipoprotein receptor-related protein), but mediates slow internalization of bound ligand.

The type-1 receptor sorLA/LR11, a member of the Vps10p-domain receptor family that also contains domains characterizing members of the LDL (low-density lipoprotein) receptor family, has been shown to induce increased uPAR (urokinase receptor) expression as well as enhanced migration and invasion activities in smooth muscle cells in the presence of PDGF-BB (platelet-derived growth factor-BB). Here we show that sorLA interacts with both components of the plasminogen activating system and PDGF-BB similarly to LRP1 (LDL receptor-related protein/alpha2-macroglobulin receptor), which is an important clearance receptor with established functions in controlling uPAR expression as well as PDGF-BB signalling. In contrast with LRP1, sorLA does not interact with alpha2-macroglobulin, which is a binding protein for several growth factors, including PDGF-BB. By using LRP1-deficient cells transfected with sorLA, we demonstrate that sorLA-bound ligand is internalized at a much lower rate than LRP1-bound ligand, and that sorLA is inefficient in regulating cell surface uPAR expression, which depends on rapid internalization of the ternary complex between urokinase-type plasminogen activator, its type-1 inhibitor, and uPAR. Thus, although overlapping with regard to binding profiles, sorLA is substantially less efficient as a clearance receptor than LRP1. We propose that sorLA can divert ligands away from LRP1 and thereby inhibit both their clearance and signalling events mediated by LRP1.

High glucose potentiates mitogenic responses of cultured ovine coronary smooth muscle cells to platelet derived growth factor and transforming growth factor-β1

Macrovascular complications in diabetes are associated with exaggerated growth responses of vascular smooth muscle cells. We studied the effect of high glucose media on the growth responses of vascular smooth muscle cells from the left anterior descending (LAD) coronary artery of young sheep. Experiments were conducted in DMEM containing 5.5 or 25 mmol/l glucose and mitogenic responses assessed by 3H-thymidine incorporation. In the absence of growth factors there was a slight and variable response to high glucose but the maximum response to platelet derived growth factor-bb (PDGF-bb) (100 ng/ml) was increased more than 2-fold. Transforming growth factor-β1 (1 ng/ml) caused a 100% increase of the PDGF-bb response in both normal and high glucose media. The acute stimulatory effect of high glucose was not affected by pre-incubation of the cells for 24 h in the high glucose medium. The mitogenic response occurring in the presence of PDGF-bb and high glucose was totally inhibited by the tyrosine kinase inhibitors (imatinib and genistein) and could not be mimicked by increasing diacylglycerol in low glucose media with the diacylglycerol kinase inhibitor, R59949. In conclusion, high glucose, per se, only very weakly stimulates smooth muscle cell growth but it interacts positively to potentiate the responses to the vascular derived growth factors PDGF and TGF-β1. The effect of high glucose is transduced via receptor tyrosine kinases and may not involve diacylglycerol that is subject to diacylglycerol kinase catabolism. The data provide explanations for the accelerated vascular smooth muscle cell proliferation in diabetes.

Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its α receptor

AbstractBasic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) modulate vascular wall cell function in vitro and angiogenesis in vivo. The aim of the current study was to determine how bovine aorta endothelial cells (BAECs) respond to the simultaneous exposure to PDGF-BB and bFGF. It was found that bFGF-dependent BAEC migration, proliferation, and differentiation into tubelike structures on reconstituted extracellular matrix (Matrigel) were inhibited by PDGF-BB. The role played by PDGF receptor α (PDGF-Rα) was investigated by selective stimulation with PDGF-AA, by blocking PDGF-BB-binding to PDGF-Rα with neomycin, or by transfecting cells with dominant-negative forms of the receptors to selectively impair either PDGF-Rα or PDGF-Rβ function. In all cases, PDGF-Rα impairment abolished the inhibitory effect of PDGF-BB on bFGF-directed BAEC migration. In addition, PDGF-Rα phosphorylation was increased in the presence of bFGF and PDGF, as compared to PDGF alone, whereas mitogen-activated protein kinase phosphorylation was decreased in the presence of PDGF-BB and bFGF compared with bFGF alone. In vivo experiments showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and that PDGF-BB inhibited bFGF-induced angiogenesis in Matrigel plugs injected subcutaneously in CD1 mice. Taken together these results show that PDGF inhibits the angiogenic properties of bFGF in vitro and in vivo, likely through PDGF-Rα stimulation.