OBJECTIVE

In response to injury, aging mediates exaggerated neointimal formation, the pathologic hallmark of obliterative vascular diseases. We assessed the development of neointima in a model of mechanical vascular injury in aging mice (18 months old) and young mice (2 months old). To investigate the mechanisms by which aging affects neointimal formation, we also carried out a set of in vitro studies to characterize the biologic properties of vascular smooth muscle cells (VSMCs) derived from aging and young mice.

METHODS

Aging and young mice were subjected to wire injury to the carotid artery. Four weeks later injured arteries were harvested, and neointimal formation was histologically assessed. The profiles of angiogenesis-related genes between aortic VSMCs derived from aging and young mice were compared with complementary DNA arrays. Expression of platelet-derived growth factor receptor-alpha (PDGFR-alpha) and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) by VSMCs were assessed. Susceptibility to apoptosis in aging and young VSMCs in response to nitric oxide and serum starvation was investigated. In addition, the level of apoptosis in neointimal VSMCs (by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay) was compared between aging and young animals.

RESULTS

When compared with young mice, aging mice exhibited exaggerated neointimal formation (intima-media ratio, 1.17 +/- 0.57 vs 0.49 +/- 0.16; P < .0001). Aging VSMCs expressed higher levels of PDGFR-alpha (12.0% +/- 2.7% vs 3.2 +/- 0.67%; P = .034) and greater proliferative response (4-fold increase) to PDGF-BB, compared with young VSMCs. However, aging VSMCs were less susceptible to apoptosis when subjected to serum starvation (75% less) and exposure to nitric oxide (50% less). Furthermore, there was more apoptosis in the neointima of young arteries than in their aging counterparts (8.75% +/- 3.3% vs 2.8% +/- 1.9; P = .021).

CONCLUSIONS

Age-dependent increases in PDGFR-alpha may alter VSMC proliferation, and when coupled with resistance to apoptosis could contribute to exaggerated neointima formation in aging animals. Of significance, our findings in the mouse will enable application of abundant molecular tools afforded by this species to further dissect the mechanisms of exaggerated neointimal formation associated with aging.

CONCLUSIONS

Neointimal formation is the pathologic hallmark of obliterative vascular diseases, including primary atherosclerosis, post stent restenosis, graft occlusion after vascular bypass procedures, and transplant allograft vasculopathy. Aging is an independent risk factor for development of cardiovascular diseases, and aging exaggerates neointimal formation after vascular injury. Understanding the mechanisms responsible for this phenomenon may facilitate prevention or provide new therapies for vascular occlusive diseases, which are so prevalent in the aging population. Our ability to reproduce the model in the mouse will no doubt facilitate such understanding.

Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if differences in primary embryonic lineage can give rise to SMCs with stable differences in growth and differentiation properties, we isolated Ect and Mes SMCs from the Day 14 chick embryo aorta. We report that despite different primary embryonic origins, Ect and Mes SMCs express nearly identical levels of seven SMC differentiation markers in vitro, consistent with their common smooth muscle developmental fates in vivo. By contrast, Ect SMCs displayed a greater capacity for growth in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell-cell interactions. Most of the peptide growth factors tested that might account for serum-independent growth (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent differences in SMC responses to transforming growth factor-beta (TGF-beta). Exposure to TGF-beta1 (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-beta1 was growth inhibitory. In SMC cultures transfected with p3TP-lux, a luciferase reporter controlled by the TGF-beta1-response elements of the human PAI-1 promoter, TGF-beta1 (120 pM) produced 12 ± 2-fold increases in luciferase activity in Ect SMCs and only 3 ± 1.5-fold increases in Mes SMCs. Analysis of TGF-beta receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-beta receptors. However, using a polyclonal antibody specific for the chick type II TGF-beta receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-beta receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent differences in growth and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and growth modifiers, e.g., the TGF-betas. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to factors involved in the progression of vascular disease later in life.

Recent studies have indicated that peroxisome proliferator-activated receptor gamma (PPARgamma) is capable of modulating inflammation, which prompted us to investigate the potential of PPARgamma ligands as lung protective agents in pulmonary fibrosis. The present study was undertaken to investigate the effects of rosiglitazone (RSG), a highly potent ligand of PPARgamma, on migration, proliferation, and phenotypic differentiation of human lung fibroblasts (MRC-5) and to explore its potential for therapy of pulmonary fibrosis. The cell migration potential was observed in a scratch wound model. Cell proliferation was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, immunocytochemical staining, and flow cytometry, and protein expression by Western blot analysis. RSG slowed cell migration distance induced by fetal bovine serum (FBS), decreased cell proliferation initiated by FBS or platelet-derived growth factor-BB (PDGF-BB), and decreased alpha-smooth muscle actin (alpha-SMA) protein expression induced by transforming growth factor-beta1 (TGF-beta1). In addition, RSG incubation reduced the ratio of phospho-extracellular signal-regulated kinases 1/2 (p-ERK1/2) to ERK1/2 expression stimulated by FBS, PDGF-BB, and TGF-beta1. These findings show that RSG treatment inhibits lung fibroblast migration and proliferation and myofibroblast transdifferentiation stimulated by FBS and growth factors in vitro, which suggests that PPARgamma agonists could antagonize pulmonary fibrosis and have potential for therapeutic application in pulmonary fibrosis.

EWS-Fli1, a fusion gene resulting from a chromosomal translocation t(11;22, q24;q12) and found in Ewing sarcoma and primitive neuroectodermal tumors, encodes a transcriptional activator and promotes cellular transformation. However, the precise biological functions of its products remain unknown. To investigate the role of EWS-Fli1 in cell growth signaling, we transfected Ewing sarcoma TC-135 cells with short interfering RNAs for EWS-Fli1. EWS-Fli1 knockdown reduced cell growth and platelet-derived growth factor (PDGF)-BB-induced activation of the growth signaling enzymes. Interestingly, phospholipase D2 (but not the PDGF-BB receptor) showed marked down-regulation in the EWS-Fli1-knocked down TC-135 cells compared with the control cells. In Ewing sarcoma TC-135 cells, the PDGF-BB-induced phosphorylation of growth signaling involving extracellular signal-regulated kinase, Akt, p70S6K, and the expression of cyclin D3 were markedly inhibited by transfection with short interfering RNA phospholipase (PL)-D2. The PDGF-BB-induced activation of growth signaling was also suppressed by 1-butanol, which prevents the production of phosphatidic acid by phospholipase D (but not by t-butyl alcohol), thereby implicating PLD2 in PDGF-BB-mediated signaling in TC-135 cells. These results suggest that EWS-Fli1 may play a role in the regulation of tumor proliferation-signaling enzymes via PLD2 expression in Ewing sarcoma cells.

Several investigations have suggested a putative tumor suppressor role for lysyl oxidase because it is down-regulated in many human and oncogene-induced tumors. To address this issue we down-regulated the enzyme in normal rat kidney fibroblasts by stable transfection of its cDNA in an antisense orientation. The selected clones revealed an absence of lysyl oxidase and dramatic phenotypic changes, interpretable as signs of transformation. The antisense lysyl oxidase clones showed, indeed, loose attachment to the plate and anchorage-independent growth and were highly tumorigenic in nude mice. Moreover, we found an impaired response of the PDGF and IGF-1 receptors to their ligands. In particular, the transformed cells showed a down-regulation of both PDGF receptors and expressed the 105-kDa isoform of the IGF-1 beta receptor, which was not present in the normal control cells. The lack of response to PDGF-BB has been described as a feature of many ras-transformed phenotypes. Therefore, we looked at the status of the p21(ras). Indeed, we found a significantly higher level of active p21(ras) both during steady-state growth and prolonged starvation. Our data reveal new evidence for a tumor suppressor activity of lysyl oxidase, highlighting its particular role in controlling Ras activation and growth factor dependence.

OBJECTIVE

To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP).

METHODS

PRP obtained from 6 horses.

METHODS

PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 μg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor β, isoform 1 (TGFβ₁); and insulin-like growth factor, isoform 1 (IGF-1) were quantified by use of ELISAs. Statistical analysis was conducted via repeated-measures ANOVA.

RESULTS

Platelet numbers were significantly higher in tube-prepared PRP than in automated-prepared PRP Growth factor concentrations did not differ significantly between preparation methods. Mean PDGF-BB concentration ranged from 134 to 7,157 pg/mL, mean TGFβ₁ concentration ranged from 1,153 to 22,677 pg/mL, and mean IGF-1 concentration ranged from 150 to 280 ng/mL. Shear force did not affect growth factor concentrations. Dose-dependent increases in PDGF-BB and TGFβ₁ were detected in response to collagen, but equalled only 10% of the estimated total platelet content. Concentrations of IGF-1 were not significantly different among treatments and negative or positive control treatments. Serum concentrations of PDGF-BB and TGFβ₁ exceeded concentrations in PRP for most treatment conditions.

CONCLUSIONS

Release of growth factors from equine PRP was negligible as a result of the injection process alone. Investigation of platelet-activation protocols is warranted to potentially enhance PRP treatment efficacy in horses.

Platelet-derived growth factor-BB (PDGF-BB) plays important roles in regenerating damaged tissue. In this study we investigated the effects of a tissue-engineered bone combined with recombinant human PDGF-BB (rhPDGF-BB), bone marrow stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) to repair critical-size calvarial bone defects in rat. Proliferation and osteogenic differentiation of BMSCs treated with different concentration rhPDGF-BB (0, 10, and 50 ng/ml) was evaluated by MTT, alkaline phosphatase (ALP) activity, alizarin red staining and real-time quantitative PCR (RT-qPCR) analysis of osteogenic gene. BMSCs were then combined with rhPDGF-BB-loaded β-TCP and transplanted into 5 mm calvarial bone defects. The new bone formation and mineralization was evaluated by micro-computerized tomography (Micro-CT) and histological analysis at week 8 after operation. It was observed that the proliferation of BMSCs treated with rhPDGF-BB was enhanced with a time- and dose- dependent manner. There were increased ALP activity, mineralized deposition and elevated mRNA levels of osteogenic gene for BMSCs treated with rhPDGF-BB, particularly in the 50 ng/ml group. Histological analysis showed new bone formation and mineralization in the rhPDGF-BB/BMSCs/β-TCP group was significantly higher than BMSCs/β-TCP, rhPDGF-BB/β-TCP, and β-TCP alone group (P < 0.05). In conclusion, rhPDGF-BB/BMSCs/β-TCP is a promising tissue-engineered bone for craniofacial bone regeneration.

OBJECTIVE

This study aimed to observe nine factors expressed in rat ischemic brain after transplantation of bone marrow stromal cells (BMSC) and/or endothelial progenitor cells (EPC). These factors were vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-l), transforming growth factor-β (TGF-β), platelet-derived growth factor-BB (PDGF-BB), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF).

METHODS

Adult Wistar rats were divided randomly into four groups: a vehicle group, BMSC group, EPC group and BMSC combined with EPC group. The rats were subjected to middle cerebral artery occlusion (MCAO) then implanted intravenously with 3 × 10(6) BMSC, EPC, BMSC/EPC or phosphate-buffered saline (PBS) 24 h after MCAO. Neurologic functional deficits were measured on days 1, 7, 14, 28 after transplantation. On day 7 after transplantation, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and Western blot were employed to detect the expression of VEGF, SDF-1, bFGF, IGF-l, TGF-β, PDGF-BB, BDNF, GDNF and NGF.

RESULTS

The neurologic evaluation found that the neurologic severity scores were no different between the four groups on day 1, and the scores of rats in the BMSC/EPC group were significantly lower than those of rats in the other groups on days 7, 14 and 28 after transplantation. The expressions of bFGF, VEGF and BNDF were significantly higher in the BMSC/EPC group compared with the other groups.

CONCLUSIONS

The intravenous transplantation of BMSC combined with EPC could promote the functional rehabilitation of rats with focal cerebral ischemia, and the mechanism may be related to the enhanced expression of factors.

An enhanced activity of receptor tyrosine kinases (RTKs), such as the platelet-derived growth factor (PDGF) alpha-receptor (PDGF-Ralpha) or the PDGF beta-receptor (PDGF-Rbeta), is involved in the development of proliferative diseases. We have previously demonstrated that green tea catechins containing a galloyl group in the third position of the catechin structure interfere with PDGF-BB-induced mitogenic signaling pathways by inhibiting tyrosine phosphorylation of the PDGF-Rbeta. However, the underlying cellular and molecular mechanisms are unknown. Using human vascular smooth muscle cells (VSMC) and porcine endothelial cells (AEC) stably transfected with PDGF-Ralpha and -beta, respectively, we demonstrate that EGCG preferably inhibited PDGF-BB isoform-mediated signal transduction pathways and cell proliferation. To elucidate cellular and molecular mechanisms of the inhibitory effects of EGCG, we studied the distribution of incorporated EGCG into cellular compartments after subcellular fractionation. Interestingly, most (85%) of the EGCG was found in the cytoplasmic fraction, whereas only ~2% was found within the cell plasma membranes. However, no alteration of membrane fluidity has been observed after treatment of VSMC with 50 microM EGCG. Binding studies with [125I]-PDGF-BB on EGCG-treated VSMC demonstrated that the specific binding of PDGF-BB was completely abolished. Moreover, when [125I]-PDGF-BB was incubated with VSMC in the presence of EGCG, a 50% reduction of cellular [125I]-PDGF-BB binding was observed. Our findings suggest that plasma membrane incorporated EGCG or soluble EGCG directly interacts with PDGF-BB, thereby preventing specific receptor binding.

Epidemiological studies suggest that green tea consumption is associated with a reduced risk of cardiovascular disease. Antioxidative properties of green tea flavonoids, catechins, have been believed to be involved in the antiatherogenic effect of green tea, since catechins inhibit low density lipoprotein oxidation. The migration of vascular smooth muscle cells (SMCs) from the tunica media to the subendothelial region is a key event in the development and progression of atherosclerosis and post-angioplasty vascular remodeling. Matrix metalloproteinases (MMPs) play a key role in these processes of SMC migration. In the present study, we investigated the effect of catechins on the gelatinolytic activity of MMP-2 that was derived from cultured bovine aortic SMCs. We also investigated the effect of catechins on the SMC invasion through the reconstituted basement membrane barrier. A major constituent of green tea catechins, (-)-epigallocatechin gallate (EGCG), inhibited the gelatinolytic activity of MMP-2 and concanavalin A (ConA)-induced pro-MMP-2 activation without the influence of membrane-type MMP expression in SMCs. EGCG also inhibited the SMC invasion through the basement membrane barrier in a concentration-dependent manner without any influence of SMC migration across the basement membrane protein thin-coated filter. The antagonistic effects of other catechins, namely (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG), on gelatinolytic activity of MMP-2, ConA-induced pro-MMP-2 activation, or PDGF-BB-directed SMC invasion were much less pronounced than those of EGCG. Also, (+)-catechin and (-)-epicatechin failed to show any effect. These findings may suggest that the anti-invasive and anti-metalloproteinase activities involve at least part of the anti-atherogenic action of catechin in accordance with the antioxidant properties of catechin.

OBJECTIVE

Systemic Sclerosis (SSc) is characterized by a microvascular damage due to an impairment of different angiogenic and angiostatic factors. Aim of this study was to measure plasma levels of nine molecules involved in these vascular processes in a group of SSc patients, respect to healthy controls (NC).

METHODS

Sixty-five patients (M/F = 2/63; mean age = 57.29 yrs; mean disease duration = 9,63 yrs) with established SSc according to ARA criteria, and sixteen age- and sex-matched NC were enrolled. Plasma levels of vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), platelet derived growth factor- bb (PDGF-BB), platelet endothelial cellular adhesion molecule-1 (PECAM-1), leptin, hepathocyte growth factor (HGF), follistatin, granulocyte-colony stimulating factor (G-CSF) and interleukin 8 (IL-8) were measured using commercially available immunoassay kits (Human Angiogenesis 9-Plex Panel, Bio-Rad Laboratories).

RESULTS

We detected a significant increase of Ang-2 (median value 1315.4 pg/ml vs 538.73 pg/ml; p=0.0292), HGF (median value 2886.16 pg/ml vs 1296.16 pg/ml; p=0.0001), IL-8 (median value 32.22 pg/ml vs 16.86 pg/ml; p=0.02), leptin (median value 32589,1 pg/mg vs 10679.61 pg/ml; p=0.0065), PDGF-BB (median value 7258.6 pg/ml vs 2913.44 pg/ml; p=0.0005), PECAM-1(median value 21681.81 pg/ml vs 10354.53 pg/ml; p=0.0003) and VEGF (median value 236.72 pg/ml vs 122.905 pg/ml; p=0.0073) in patients with SSc respect to NC. Higher levels of PDGF-BB (p=0.03) and PECAM-1 (p=0.05) were found in patients with digital ulcers while lower levels of PECAM-1 were found in patients with pulmonary hypertension (PH). Besides levels of IL-8 were higher in patients with PH (p=0.04) and lower in those with pulmonary fibrosis (p=0.5), while levels of Ang-2 were higher in those with a 'late' nailfold video-capillaroscopy (NVC) pattern respect to those with an 'early/active' one (p=0.05). Moreover, plasma levels of VEGF (p=0.02) and PDGF-BB (p= 0.04) were significantly higher in those patients positive for anti-topoisomerase 1 antibodies.

CONCLUSIONS

Our findings show significantly higher circulating levels of seven angiogenic parameters in SSc patients, thus reflecting the disregulation of endothelium in this disease. Abnormal levels of these molecules may be considered an attempt for compensatory although ineffective mechanisms of vascular function, leading to the development of the main clinical manifestations of SSc.

Platelet-derived growth factor (PDGF) isoforms regulate cell proliferation, migration and differentiation both in embryonic development and adult tissue remodeling. At the cellular level, growth-factor signaling is often modulated by endocytosis. Despite important functions of PDGF, its endocytosis remains poorly studied, mainly for lack of tools to track internalized ligand by microscopy. Here, we developed such a tool and quantitatively analyzed internalization and endosomal trafficking of PDGF-BB in human fibroblasts. We further show that PDGF can be internalized in the presence of dynamin inhibitors, arguing that both dynamin-dependent and dynamin-independent pathways can mediate PDGF uptake. Although these routes operate with somewhat different kinetics, they both ultimately lead to lysosomal degradation of PDGF. Although acute inhibition of dynamin activity only moderately affects PDGF endocytosis, it specifically decreases downstream signaling of PDGF via signal transducer and activator of transcription 3 (STAT3). This correlates with reduced expression of MYC and impaired cell entry into S-phase, indicating that dynamin activity is required for PDGF-induced mitogenesis. Our data support a general view that the components governing endocytic trafficking may selectively regulate certain signaling effectors activated by a growth factor.

Retinal gliosis is characterized by biochemical and physiological changes that often lead to Müller glia proliferation and hypertrophy and is a feature of many neuro-degenerative and inflammatory diseases such as proliferative vitreoretinopathy (PVR). Although Müller glia are known to release inflammatory factors and cytokines, it is not clear whether cytokine production by these cells mirrors the pattern of factors present in the gliotic retina. Lysates from normal cadaveric retina and gliotic retinal specimens from patients undergoing retinectomy for treatment of PVR, the Müller cell line MIO-M1 and four human Müller glial cell preparations isolated from normal retina were examined for their expression of cytokines and inflammatory factors using semi-quantitative dot blot antibody arrays and quantitative arrays. Comparative analysis of the expression of inflammatory factors showed that in comparison with normal retina, gliotic retina exhibited greater than twofold increase in 24/102 factors examined by semiquantitative arrays, and a significant increase in 19 out of 27 factors assessed by quantitative methods (P < 0.05 to P < 0.001). It was observed that with the exception of some chemotactic factors, the majority of cytokines and inflammatory factors were produced by Müller glia in vitro and included G-CSF, MCP-1, PDGF-bb, RANTES, VEGF, and TGFβ2. These results showed that a large number of inflammatory factors expressed by Müller glia in vitro are upregulated in the gliotic retina, suggesting that targeting the production of inflammatory factors by Müller glia may constitute a valid approach to prevent neural damage during retinal gliosis and this merits further investigations.

The repair of nerve gap injuries with tubular nerve guides has been used extensively as an in vivo test model in identifying substances which may enhance nerve regeneration. The model has also been used clinical nerve repair. The objective of this study was to compare three different gel matrix-forming materials as potential vehicles for growth factors in this system. The vehicles included a laminin containing extracellular matrix preparation (Biomatrix), collagen, and a 2% methylcellulose gel. The growth factor test substance consisted of a combination of platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor I (IGF-I). An 8-mm gap in rat sciatic nerve was repaired with a silicone tube containing each of the vehicles alone or with a combination of each vehicle plus PDGF-BB and IGF-I. At 4 weeks after injury, the application of the growth factor combination significantly stimulated axonal regeneration when applied in methylcellulose or collagen, but not in Biomatrix. A similar trend was present between the vehicle control groups. By 8 weeks after injury, nerves repaired with methylcellulose as a vehicle had significantly greater conduction velocity than either collagen or Biomatrix. It was concluded that a 2% methylcellulose gel was the best of the three matrices tested, both in its effects on nerve regeneration and flexibility of formulation.

Bone has a high capacity for self-renewal and repair. Prolonged local secretion of interleukin 1β (IL-1β), however, is known to be associated with severe bone loss and delayed fracture healing. Since induction of bone resorption by IL-1β may not sufficiently explain these pathologic processes, we investigated, in vitro, if and how IL-1β affects migration of multipotent mesenchymal stromal cells (MSC) or osteoblasts. We found that homogenous exposure to IL-1β significantly diminished both nondirectional migration and site-directed migration toward the chemotactic factors platelet-derived growth factor (PDGF)-BB and insulin like growth factor 1 (IGF-1) in osteoblasts. Exposure to a concentration gradient of IL-1β induced an even stronger inhibition of migration and completely abolished the migratory response of osteoblasts toward PDGF-BB, IGF-1, vascular endothelial growth factor A (VEGF-A) and the complement factor C5a. IL-1β induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK) activation and inhibition of these signaling pathways suggested an involvement in the IL-1β effects on osteoblast migration. In contrast, basal migration of MSC and their migratory activity toward PDGF-BB was found to be unaffected by IL-1β. These results indicate that the presence of IL-1β leads to impaired recruitment of osteoblasts which might influence early stages of fracture healing and could have pathological relevance for bone remodeling in inflammatory bone disease.

The development of an intimal proliferative lesion after balloon catheter de-endothelialization was studied in congenitally athymic nude rats lacking T lymphocytes. Significant intimal thickening was observed in both the homozygous (nu/nu) and euthymic heterozygous (nu/+) animals 6 days after injury, which increased further after 10 days. There was no significant difference in mean intimal:medial cross-sectional area between the nu/nu and nu/+ animals at either time. Approximately 1% of the cells in the neointima of both groups of animals were leukocytes (OX-1 positive); 0.7% were macrophages (ED-1 positive). In neither nu/nu nor nu/+ animals did T lymphocytes (OX-19-positive cells) constitute more than 0.1% of the neointimal cell population. These data suggest that T lymphocytes do not play a significant role in the accumulation of neointimal cells. The presence of macrophages within the lesions raises the possibility that they may be involved in the recruitment and proliferation of smooth muscle cells. In vitro characterization of nu/nu carotid medial smooth muscle cells demonstrated approximately 500,000 binding sites for platelet-derived growth factor (PDGF)-BB and few PDGF-AA binding sites (less than 10,000). The mitogenic and chemotactic responses of these cells to the three dimeric forms of PDGF correlated with this receptor subunit distribution. Platelet-derived growth factor accounted for approximately 50% of the mitogenic activity of a rat platelet releasate. Platelet-derived growth factor-BB and PDGF-AB were both potent chemotactic agents for the nude rat carotid smooth muscle cells with a peak response at approximately 10 ng/ml. In contrast, PDGF-AA, transforming growth factor beta, and basic fibroblast growth factor were only weak chemoattractants for these cells.

Underlying the dynamic regulation of tropoelastin expression and elastin formation in development and disease are transcriptional and post-transcriptional mechanisms that have been the focus of much research. Of particular importance is the cytokine-governed elastin regulatory axis in which the pro-elastogenic activities of transforming growth factor β-1 (TGFβ1) and insulin-like growth factor-I (IGF-I) are opposed by anti-elastogenic activities of basic fibroblast growth factor (bFGF/FGF-2), heparin-binding epidermal growth factor-like growth factor (HB-EGF), EGF, PDGF-BB, TGFα, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β and noncanonical TGFβ1 signaling. A key mechanistic feature of the regulatory axis is that cytokines influence elastin formation through effects on the cell cycle involving control of cyclin-cyclin dependent kinase complexes and activation of the Ras/MEK/ERK signaling pathway. In this article we provide an overview of the major cytokines/growth factors that modulate elastogenesis and describe the underlying molecular mechanisms for their action on elastin production.

OBJECTIVE

To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes.

METHODS

An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-beta2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of alpha-smooth muscle actin (alphaSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes.

RESULTS

Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-beta2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at approximately 4 hours after stimulation (180.8%, P <0.01), TGF-beta2-elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P <0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03-20.00 microM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-beta2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with alphaSMA overexpression, which was downregulated by hydroxyfasudil in part (P <0.01).

CONCLUSIONS

The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-beta2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rho-kinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-beta2-dependent contractile properties of hyalocytes.

Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRβ within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

The abilities of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) to regulate cAMP metabolism and mitogen-activated protein kinase (MAP kinase) activity were compared in human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP accumulation up to 150-fold in a concentration-dependent manner (EC50 approximately 0.7 nM). The peak of cAMP formation and cAMP-dependent protein kinase (PKA) activity occurred approximately 5 min after the addition of PDGF and rapidly declined thereafter. Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA activity by an additional 2.5-3-fold, whereas IBMX alone was essentially without effect. The PDGF-stimulated increase in cAMP was prevented by addition of the cyclooxygenase inhibitor indomethacin, consistent with release of prostaglandins stimulating cAMP. PDGF, but not IGF-I, stimulated MAPK activity, cytosolic phospholipase A2 (cPLA2) phosphorylation, and cAMP synthesis which indicated a key role for MAP kinase in the activation of cPLA2. Further, PDGF stimulated the rapid release of arachidonic acid and synthesis of prostaglandin E2 (PGE2) which could be inhibited by a cPLA2 inhibitor (AACOCF3). Calcium mobilization was required for PDGF-induced arachidonic acid release and PGE2 synthesis but not for MAPK activation, whereas PKC was required for PGE2-mediated activation of PKA. In summary, these results demonstrated that PDGF increases cAMP formation and PKA activity through a MAP kinase-mediated activation of cPLA2, arachidonic acid release, and PGE2 synthesis in human arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) stimulates tumor growth and progression by affecting tumor and stromal cells. In the HaCaT skin carcinogenesis model, transfection of immortal nontumorigenic and PDGF-receptor-negative HaCaT keratinocytes with PDGF-B induced formation of benign tumors. Here, we present potential mechanisms underlying this tumorigenic conversion. In vivo, persistent PDGF-B expression induced enhanced tumor cell proliferation but only transiently stimulated stromal cell proliferation and angiogenesis. In vitro and in vivo studies identified fibroblasts as PDGF target cells essential for mediating transient angiogenesis and persistent epithelial hyperproliferation. In fibroblast cultures, long-term PDGF-BB treatment caused an initial up-regulation of vascular endothelial growth factor (VEGF)-A, followed by a drastic VEGF down-regulation and myofibroblast differentiation. Accordingly, in HaCaT/PDGF-B transplants, initially enhanced VEGF expression by stromal fibroblasts was subsequently reduced, followed by down-regulation of angiogenesis, myofibroblast accumulation, and vessel maturation. The PDGF-induced, persistently increased expression of the hepatocyte growth factor by fibroblasts in vitro and in vivo was most probably responsible for enhanced epithelial cell proliferation and benign tumor formation. Thus, by paracrine stimulation of the stroma, PDGF-BB induced epithelial hyperproliferation, thereby promoting tumorigenicity, whereas the time-limited activation of the stroma followed by stromal maturation provides a possible explanation for the benign tumor phenotype.

OBJECTIVE

Here we investigated the role of spleen tyrosine kinase (Syk) in the migration induced by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells (RASMC).

METHODS

Cell migration was determined using a Boyden chamber, by wound-healing, and by aortic ring assays. Activity of Syk, mitogen-activated protein kinase (MAPK), and heat shock protein 27 (HSP27) were tested using immunoblotting with kinase inhibitors and small interference RNAs.

RESULTS

PDGF-BB induced binding of Syk to the PDGFbeta receptor and increased the phosphorylation of Syk and migration in RASMC. These effects of PDGF-BB were inhibited by piceatannol, an inhibitor of Syk. PDGF-BB increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and HSP27, which were significantly inhibited by piceatannol and in Syk-knockdown cells. The p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 inhibited the migration, which was further inhibited by the combination of these inhibitors. SB203580, but not PD98059, inhibited the phosphorylation of HSP27 induced by PDGF-BB in RASMC. PDGF-BB-induced migration was attenuated in HSP27-knockdown cells. Kinase inhibitors and Syk-knockdown diminished PDGF-BB-induced sprout outgrowth in the aortic ring assay.

CONCLUSIONS

These results imply that Syk is an upstream signal of the p38 MAPK/HSP27 and ERK1/2 pathways that contributes to PDGF-BB-mediated migration in RASMC.

Endoplasmic reticulum (ER) stress and inappropriate adaptation through the unfolded protein response (UPR) are predominant features of pathological processes. However, little is known about the link between ER stress and endovascular injury. We investigated the involvement of ER stress in neointima hyperplasia after vascular injury. The femoral arteries of 7-8-week-old male mice were subjected to wire-induced vascular injury. After 4 weeks, immunohistological analysis showed that ER stress markers were upregulated in the hyperplastic neointima. Neointima formation was increased by 54.8% in X-box binding protein-1 (XBP1) heterozygous mice, a model of compromised UPR. Knockdown of Xbp1 in human coronary artery smooth muscle cells (CASMC) in vitro promoted cell proliferation and migration. Furthermore, treatment with ER stress reducers, 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), decreased the intima-to-media ratio after wire injury by 50.0% and 72.8%, respectively. Chronic stimulation of CASMC with PDGF-BB activated the UPR, and treatment with 4-PBA and TUDCA significantly suppressed the PDGF-BB-induced ER stress markers in CASMC and the proliferation and migration of CASMC. In conclusion, increased ER stress contributes to neointima formation after vascular injury, while UPR signaling downstream of XBP1 plays a suppressive role. Suppression of ER stress would be a novel strategy against post-angioplasty vascular restenosis.