OBJECTIVE

To document the expression patterns of various matrixins, cytokines and angiogenic factors in plasma to assess their involvement in the pathogenesis of moyamoya disease (MMD).

METHODS

This study included plasma samples from 20 MMD patients and nine healthy individuals. The plasma concentration of five matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-12), monocyte chemoattractant protein-1 (MCP-1), resistin, three interleukins (IL-1beta, IL-6, IL-8), tumour necrosis factor-alpha, vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor was determined using multianalyte profiling systems. The concentration of the tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) was measured using ELISA. Gelatin zymography for MMP-2 and MMP-9 was also performed.

RESULTS

MMD patients exhibited significantly higher plasma concentrations of MMP-9, MCP-1, IL-1beta, VEGF and PDGF-BB, and lower plasma concentrations of MMP-3, TIMP-1 and TIMP-2 compared with healthy controls. Significant correlations were found among MMP-9, MCP-1, VEGF, PDGF-BB and TIMP-2 in MMD patients.

CONCLUSIONS

There were distinctive expression patterns of matrixins, cytokines and angiogenic factors in MMD patients, which seemed to correlate with disease pathogenesis. The balance between MMPs and TIMPs was disrupted in MMD and correlated with disease pathogenesis. Increased plasma levels of MCP-1 and VEGF in MMD patients may play a role in the recruitment of vascular progenitor cells and in the formation of collateral vessels.

Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKCalpha and PKCbetaII levels were increased in 25 mmol/L glucose. However, only PKCbetaII inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKCbetaII were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKCbetaII inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKCbetaII was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKCbetaII-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

Platelet concentrates (PCs) constitute new biological mediators used in osseous reconstructive surgery. In this study, we assessed (i) the effects of various concentrations of calcium and thrombin on the kinetics of platelet-derived growth factor (PDGF-BB), transforming growth factor-beta1(TGF-beta 1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) release by PCs and (ii) the contribution of PC supernatants to endothelial cell proliferation. Our results indicate that high concentrations of calcium (Ca) and thrombin (Thr) trigger an immediate and significant increase in bFGF, TGF-beta 1 and PDGF-BB concentrations. Thereafter, PDGF-BB, VEGF and TGF-beta 1 levels remained generally constant over a 6-day period while a decrease in bFGF concentrations was noted after 24h. Lower Ca and Thr concentrations tended to reduce and delay growth factors release from PCs. Endothelial cell proliferation was greatly enhanced with PC supernatants (mean: 20-fold increase). This was especially evident when endothelial cells were treated with supernatants harvested early after PC treatment with high concentrations of Ca and Thr or later after PC treatment with low Ca and Thr concentrations. Additional research aiming to measure the effects of Ca and Thr on bone formation in vivo is needed.

BACKGROUND

Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis.

OBJECTIVE

To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.

RESULTS

We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury.

CONCLUSIONS

We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet-rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF-β1, TGF-β2, EGF, IGF-1, PDGF-AB, PDGF-BB, and VEGF) and anti-inflammatory (IL-1ra, sTNF-RI, sTNF-RII, IL-4, IL-10, IL-13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL-1α, IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18). Human articular chondrocytes were pre-incubated with the antagonists IL-1ra, sTNF-RI, or APS prior to the addition of recombinant human IL-1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP-13 in the culture medium were evaluated by ELISA. MMP-13 production stimulated in chondrocytes by IL-1β or TNFα was reduced by rhIL-1ra and sTNF-RI to near basal levels. APS was also capable of inhibiting the production of MMP-13 induced by both IL-1β and TNFα. The combination of anabolic and anti-inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA.

Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels ofplatelet-derived growth factor alpha-receptor and beta-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor alpha-receptor or beta-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers.

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

BACKGROUND

The elucidation of molecular mechanisms of vascular cell biology has markedly influenced our thinking on the pathophysiology of vascular disease. Antisense oligonucleotide gene therapy has helped identify proteins critical to cell-cycle progression and proliferation and possible therapeutic strategies to combat human disease. This approach, however, has not yet been used to examine the contribution of chemotactic proteins and/or their receptors. Platelet-derived growth factor-BB (PDGF-BB) released from activated platelets adherent to subendothelial connective tissue is a principal smooth muscle cell chemotactic factor.

RESULTS

A series of experiments was performed to assess the capacity of antisense oligonucleotides to reduce PDGF-beta receptor subunit (PDGFR-beta) expression and the contribution of PDGFR-beta in neointimal formation. Sustained, direct, and local perivascular administration of two different antisense oligonucleotide sequences complementary to PDGFR-beta mRNA almost completely abolished the expression of PDGFR-beta protein in the intima and media of injured carotid arteries and decreased neointimal formation by 80% and 60%, respectively. Furthermore, neointimal formation correlated precisely with PDGFR-beta expression in an exponential fashion.

CONCLUSIONS

Thus, myointimal proliferation depends on both PDGFR-beta overexpression and its activation by PDGF-BB. Removal of either of these two elements can suppress neointimal formation.

OBJECTIVE

While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth.

METHODS

Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein.

RESULTS

In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR.

CONCLUSIONS

Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.

Treatment of Swiss 3T3 cells with a subsaturating concentration of recombinant Pasteurella multocida toxin (rPMT) markedly potentiated the production of inositol phosphates induced by bombesin, vasopressin, and endothelin but not by platelet-derived growth factor (PDGF) (AA and BB homodimers). Similarly, the neuropeptides but not PDGF caused a shift in the dose-dependent increase in inositol phosphates induced by rPMT. The rate of accumulation of inositol phosphates induced by bombesin was increased 2-fold by rPMT treatment while that of PDGF was unaffected. rPMT treatment also enhanced bombesin-induced inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase C gamma. Depletion of protein kinase C increased rPMT-induced inositol phosphates in a manner similar to that observed for bombesin but not PDGF. Thus, rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis persisted in streptolysin O-permeabilized cells. Addition of guanosine 5'-O-(beta-thiodiphosphate) to permeabilized cells markedly reduced rPMT-induced inositol phosphates in a time- and dose-dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. Our results strongly suggest that the action of rPMT facilitates the coupling of G protein to phospholipase C.

The response of rat aortic smooth muscle cells to all three isoforms of platelet-derived growth factor (PDGF) was studied. 5,000 binding sites/cell were estimated for rPDGF-AA (Kd 0.22 nM), 45,000 for rPDGF-AB and (Kd 0.4 nM), and 31,000 for rPDGF-BB (Kd 0.29 nM). rPDGF-AB and -BB stimulated effectively [3H]thymidine incorporation, inositol 1,4,5-trisphosphate release, diacylglycerol productions, [Ca2+]i increase, and pHi changes at concentration in the range from 3 to 10 ng/ml. The extent of DNA synthesis stimulated by rPDGF-AA varied considerably, and in all cases higher concentrations than 10 ng/ml were required. rPDGF-AA did not stimulate inositol-1,4,5-trisphosphate release, [Ca2+]i increase or pHi changes but induced the production of diacylglycerol, although with a different kinetic compared with that observed with rPDGF-AB or -BB. Apparently rPDGF-AA acts via a different mechanism, generating diacylglycerol without the release of inositol-1,4,5-trisphosphate.

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate M(r) 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4 kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non- or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

The effects of 5 different growth factors [EGF, PDGF(bb), TGF-alpha, bFGF and IL-2] were studied on tumour spheroids obtained from 5 different human glioma cell lines (U-251MG, D-263MG, D-37MG, D-54MG, GaMG). The expression of EGF and PDGF receptors as well as the endogenous production of TGF-alpha and PDGF were studied by Northern blot analyses. After growth-factor-exposure, tumour spheroid volume growth, and directional cell migration from the spheroids were studied. In addition, tumour-cell invasion was studied in vitro, where foetal rat-brain aggregates were used as a target for the tumour cells. In all the assays a common stimulator for most of the cell lines was EGF. The other growth factors had a more heterogeneous stimulatory effect. Tumour-cell invasion, cell growth and cell migration are biological properties which are not necessarily related to each other. This may explain why the tumours often responded differently to the growth factors in the various assay systems. Two of the cell lines studied were non-invasive (U-251MG, D-263MG). It is shown that these were stimulated both in the directional migration assay and in the spheroid-volume-growth assay. However, their non-invasive behaviour was not influenced by the growth factors studied.

Stromal cell-derived Factor-1alpha (SDF-1alpha) stimulates the migration of bone marrow (BM) cells, similar to vascular endothelial growth factor (VEGF). We previously demonstrated that inhibition of VEGF(165) by small interfering RNA inhibited Ewing's sarcoma tumor growth, tumor vessel formation and recruitment of BM cells to the tumor. To determine the importance of BM cells in tumor vessel development, we investigated the effects of SDF-1alpha on VEGF-inhibited TC/siVEGF(7-1) Ewing's tumor neovasculature formation and growth. The effect of SDF-1alpha on CD34(+) progenitor cell chemotaxis was determined in vivo. Using a BM transplantation model with GFP(+) transgenic mice as BM donors and nude mice as recipients, we evaluated the effect of SDF-1alpha on the recruitment of BM-derived cells to VEGF(165)-inhibited TC/siVEGF(7-1) tumors, as well as its effect on neovasculature development, vessel morphology and tumor growth. SDF-1alpha stimulated the migration of CD34(+) progenitor cells to Matrigel plugs in vivo and promoted the retainment of BM-derived pericytes in close association with perfused, functional tumor vessels. Intratumor inoculation of Ad-SDF-1alpha into TC/siVEGF(7-1) tumors resulted in increased SDF-1 and PDGF-BB expression, augmented tumor growth, an increase in the number of large, lumen-bearing vascular structures, and enhanced vessel pericyte coverage, with no change in VEGF(165). SDF-1alpha stimulates BM cell chemotaxis and the association of these cells with functional tumor vessels. Furthermore, SDF-1alpha enhances tumor neovascularization and growth with no alteration in VEGF(165). Our work suggests that SDF-1-mediated vasculogenesis may represent an alternate pathway that could potentially be utilized by tumors to sustain growth and neovasculature expansion after anti-VEGF therapy.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and alpha-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-β1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100×10(6) of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.

OBJECTIVE

Platelet-derived growth factor (PDGF) has been implicated in vascular proliferative retinopathies, such as diabetic retinopathy, and in nonvascular retinopathies, such as proliferative vitreoretinopathy. Traction retinal detachment is a central feature of both types of disease. Hemizygous rhodopsin promoter/PDGF-B (rho/PDGF-B) transgenic mice exhibit proliferation of vascular cells, glia, and retinal pigmented epithelial (RPE) cells, resulting in traction retinal detachment. Hemizygous rho/PDGF-A transgenic mice show mild proliferation of glial cells and no traction retinal detachments. This study was undertaken to determine whether higher levels of endogenously produced PDGF-A in the retinas of mice result in retinal detachment.

METHODS

To achieve high-level expression of PDGF-A in the retina, homozygous rho/PDGF-A (rho/PDGF-AA) mice were generated. The phenotype of these mice was compared with that of homozygous rho/PDGF-B (rho/PDGF-BB) mice and double hemizygous rho/PDGF-B-rho/PDGF-A (rho/PDGF-AB) mice.

RESULTS

Rho/PDGF-BB and rho/PDGF-AB mice showed a phenotype similar to that previously described in rho/PDGF-B mice. There was extensive proliferation of glial and vascular cells, resulting in fibrovascular membranes that detached the retina. PDGF-AA mice showed extensive proliferation of glial cells and traction retinal detachment.

CONCLUSIONS

High retinal expression of PDGF-A results in extensive proliferation of glial cells and traction retinal detachment without vascular cell involvement, similar to proliferative vitreoretinopathy in humans. High retinal expression of PDGF-B results in traction retinal detachment from proliferation of both vascular and nonvascular cells, similar to diabetic retinopathy in humans.

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.

Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1-an inhibitor of mitochondrial fission-inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders.

BACKGROUND

Platelet-derived growth factor (PDGF) has been shown in vivo to increase bone formation and supplement fracture healing, and may have a role as a therapeutic agent in the treatment of bone loss and fracture healing in humans.

OBJECTIVE

A comprehensive review of the recent literature on the effect of PDGF on bone mineral density and fracture healing.

METHODS

In vitro and in vivo evidence was systematically collected using medical search engines MEDLINE/OVID (1950 to March 2008) and EMBASE (1980 to March 2008) databases.

CONCLUSIONS

Evidence to date suggests that PDGF-BB, and to a lesser extent PDGF-AA, may have potential therapeutic use in the treatment of osteoporosis and bone healing in humans. Additionally, by targeting alpha-receptors on osteoblasts, a potential anabolic effect on bone metabolism in humans can be anticipated; however, more research needs to be done to assess the role of beta-receptors in human bone.