In addition to their role in cytokine gene regulation in T cells, nuclear factors of activated T cells (NFATs) have been shown to be involved in cardiac development and hypertrophy. We have reported previously that NFATs play an important role in the regulation of vascular smooth muscle cell (VSMC) proliferation by receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists, platelet-derived growth factor-BB (PDGF-BB) and thrombin, respectively. To understand the role of NFATs in vascular disease and development, we have now studied the role of these transcriptional factors in VSMC motility. PDGF-BB and thrombin induced VSMC motility in a dose-dependent manner. Blockade of NFAT activation resulted in substantial reduction in PDGF-BB- and thrombin-induced VSMC motility. PDGF-BB and thrombin also induced interleukin-6 (IL-6) expression in NFAT-dependent manner. Furthermore, IL-6 dose-dependently caused VSMC motility. A neutralizing anti-rat IL-6 antibody inhibited VSMC motility induced by IL-6, PDGF-BB, and thrombin. In addition, exogenous addition of IL-6 rescued both PDGF-BB- and thrombin-induced VSMC motility from inhibition by the blockade of NFAT activation. Together, these results for the first time demonstrate that NFATs mediate both RTK and GPCR agonist-induced VSMC motility via induction of expression of IL-6.

The potential use of autologous thrombocytic growth factors to accelerate bone regeneration requires improved methods of isolating platelet-rich plasma (PRP). In addition to discontinuous cell separation, a second method by which PRP is produced at the point-of-care has now become available. In this study, growth factor levels in PRP from these two sources were compared. Whole blood was drawn from 115 healthy donors (73 males, 42 females) aged 21 - 62 years (mean 36, SD 10). The PRP was separated by the blood bank (BB) using the discontinuous cell separation method or at the 'point-of-care' by the so-called 'buffy coat' method (analogous to the Curasan PRP Kit). Growth factor content differed significantly for TGF-beta1 (BB 268.65+/-70.77 ng/ml, Curasan 95.02+/-60.67 ng/ml (sign test P<0.001)) and PDGF-AB (BB 133.59+/-46.26 ng/ml, Curasan 233.70+/-111.86 ng/ml (P<0.001)), while the content of IGF-I (BB 85.37+/-25.58 ng/ml, Curasan 101.72+/-47.7 ng/ml (P<0.160)) showed no significant difference. The higher thrombocyte count in the BB PRP (BB 1434300+/-351960/ microl, Curasan 908.500+/-492.30/microl) seems to result in higher TGF-beta1 levels, while the higher leukocyte count in the Curasan PRP (BB 160+/-320/ microl, Curasan 30130+/-12500/microl) seems to result in higher PDGF-AB levels. The similar IGF-I levels in the two preparations might merely reflect similar amounts of plasma in the PRP produced by each approach.

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

The activation of the microvascular endothelial cell platelet-derived growth factor (PDGF) receptor (PDGF-R) by PDGF has been implicated in neoplastic angiogenesis. Here, we established cultures of murine bone microvascular endothelial cells and examined their response to stimulation with PDGF BB ligand and to blockade of PDGF-R signaling with the tyrosine kinase inhibitor STI571 (Gleevec). The addition of STI571 to cultures of bone endothelial cells blocked PDGF BB-induced phosphorylation in a dose-dependent manner and completely abrogated the activation of downstream targets Akt and ERK1/2. Coadministration of STI571 and Taxol also induced the activation of procaspase-3 and significant apoptosis. These data suggest that phosphorylation of PDGF-R stimulates survival pathways in bone endothelial cells and that by selectively inhibiting PDGF-R signaling with STI571, the cells are rendered sensitive to Taxol treatment. The therapeutic combination of STI571 and Taxol may be a powerful tool for targeting tumor-associated endothelial cells in the skeletal compartment.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

Chronic lung disease of prematurity (bronchopulmonary dysplasia; BPD) is characterized by an arrest in lung development. We hypothesized that early alterations in pulmonary expression of growth factors important for normal lung development would precede development of BPD. Bronchoalveolar lavage fluid (BALF) was obtained from ventilated preterm infants (n = 62) on postnatal d 0, 1, 3, and 7 and analyzed for total phospholipids (PL), VEGF, PDGF-BB, TGF-alpha and -beta1, granulocyte macrophage colony stimulating factor (GM-CSF), and keratinocyte growth factor (KGF). Levels (Ln transformed) were compared between infants developing BPD and BPD-free survivors, adjusted for potential confounders. BPD was associated with higher overall GM-CSF (beta (95% CI) = 0.69 (0.13;1.25); p < 0.05), lower overall latent TGF-beta1 (beta (95% CI) = -1.19 (-1.87, -0.39); p < 0.01) and total PL (beta (95% CI) = -0.64 (-1.23, -0.05); p < 0.05), and lower d 0 and 3 levels of VEGF (mean difference (95% CI) = -1.75 (-2.72, -0.77), p < 0.001; and -1.18 (-2.30, -0.06), p < 0.05, respectively) and TGF-alpha (mean difference (95% CI) = -0.73 (-1.42, -0.04), p < 0.05; and -1.01 (-1.64, -0.38), p < 0.01, respectively). Day 0 VEGF levels had the highest predictive value for BPD (area under receiver operating characteristic curve = 0.87; p < 0.01). In conclusion, substantial alterations in BALF growth factor levels are present in infants developing BPD. An early imbalance in pulmonary growth factors may contribute to the developmental arrest of the lung seen in BPD.

BACKGROUND

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The migration and proliferation of vascular smooth muscle cells (VSMCs) and formation of intravascular thrombosis play crucial roles in the development of atherosclerotic lesions. This study examined the effects of protocatechuic aldehyde (PCA), a compound isolated from the aqueous extract of the root of Salvia miltiorrhiza, an herb used in traditional Chinese medicine to treat a variety of vascular diseases, on the migration and proliferation of VSMCs and platelets due to platelet-derived growth factor (PDGF). DNA 5-bromo-2'-deoxy-uridine (BrdU) incorporation and wound-healing assays indicated that PCA significantly attenuated PDGF-induced proliferation and migration of VSMCs at a pharmacologically relevant concentration (100 μM). On a molecular level, we observed down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways, both of which regulate key enzymes associated with migration and proliferation. We also found that PCA induced S-phase arrest of the VSMC cell cycle and suppressed cyclin D2 expression. In addition, PCA inhibited PDGF-BB-stimulated reactive oxygen species production in VSMCs, indicating that PCA's antioxidant properties may contribute to its suppression of PDGF-induced migration and proliferation in VSMCs. Finally, PCA exhibited an anti-thrombotic effect related to its inhibition of platelet aggregation, confirmed with an aggregometer. Together, these findings suggest a potential therapeutic role of PCA in the treatment of atherosclerosis and angioplasty-induced vascular restenosis.

BACKGROUND

Clinical evidence suggests that platelet concentrate (PC) could have beneficial therapeutic effects on hard and soft tissue healing, due to the contents of growth factors (GFs) stored in the platelets. The objectives of this study were: 1) to determine the concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) released from PCs and whole blood (WB), before and after the addition of various concentrations of calcium and thrombin, and 2) to assess the physiological importance of the released GFs on angiogenesis.

METHODS

WB and PCs were harvested and prepared from three healthy volunteers. Enzyme-linked immunosorbent assay tests, specific for PDGF-BB, TGF-beta1, VEGF, and bFGF, were performed on WB and PC supernatants, collected before and 30 minutes after the addition of various concentrations of calcium and thrombin. The supernatants were also added to human umbilical vein endothelial cell (HUVEC) cultures in order to measure their effects on endothelial cell proliferation.

RESULTS

Growth factor concentrations detected in PC supernatants were significantly greater (280% to 800% increase) than concentrations present in WB supernatants. Calcium and thrombin induced immediate GF release from PCs in a dose-dependent fashion. Furthermore, PC supernatants led to greater HUVEC proliferation rates than WB supernatants. However, there was no correlation between the concentrations of specific GFs and HUVEC proliferation rates.

CONCLUSIONS

These results suggest that PCs could stimulate blood vessel formation. They also reinforce the relevance for using PCs in regenerative therapies.

Interstitial fibrosis is a marker of progression of renal impairment in diabetic nephropathy. Transforming growth factor (TGF)-beta 1 is one of a group of pro-fibrotic cytokines and growth factors that have been associated with the development of interstitial fibrosis. We have examined the modulating influence of glucose on the production of TGF-beta 1 by cultured human proximal tubular cells. Incubation of growth-arrested human proximal tubular cells (HPTC) (72 hours in serum free medium) in 25 mmol/L D-glucose resulted in increased expression of TGF-beta 1 mRNA (as assessed by reverse transcription polymerase chain reaction). This was apparent after 6 hours and increased up to 120 hours exposure. TGF-beta 1 secretion, however, as measured by specific enzyme-linked immunoassay, was unaffected by exposure to 25 mmol/L D-glucose. Sequential stimulation of HPTC, first with 25 mmol/L D-glucose for 48 hours and then with platelet-derived growth factor (PDGF) isoforms, resulted in a dose-dependent secretion of TGF-beta 1. Pre-exposure to 5 mmol/L D-glucose or 25 mmol/L L-glucose did not prime for TGF-beta 1 release. At 50 ng/ml PDGF this effect was greatest for the AA isoform (AA 31.4 +/- 7.1, AB 20.98 +/- 8.9, BB 7.8 +/- 2.2, P < 0.05 for all versus control, n = 3, mean +/- SEM ng/10(6) cells/24 hours). These effects were blocked by the addition of antibody to the PDGF alpha-receptor. TGF-beta 1 secretion was inhibited in a dose-dependent manner by pretreatment with cyclohexamide, but was not affected by pretreatment with actinomycin D. Stimulation of HPTC with a single dose of PDGF induced TGF-beta 1 mRNA; however, only after application of a second dose of PDGF (after TGF-beta 1 mRNA induction) did TGF-beta 1 protein secretion occur. We also demonstrated that PDGF stimulation of HPTC induced an inherently more stable TGF-beta 1 mRNA transcript. These findings demonstrate that elevated D-glucose concentration alone is insufficient to lead to increased TGF-beta 1 secretion by HPTC despite increased mRNA expression. However, application of a second stimulus such as PDGF, when TGF-beta 1 mRNA expression is increased, leads to increased protein synthesis and secretion of TGF-beta 1. This implies that elevated glucose concentrations might prime proximal tubular cells for TGF-beta 1 synthesis and thus contribute to the development of interstitial fibrosis.

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.

Platelet-derived growth factor (PDGF) exerts multiple cellular effects that stimulate wound repair in multiple tissues. However, a major obstacle for its successful clinical application is the delivery system, which ultimately controls the in vivo release rate of PDGF. Polylactic-co-glycolic acid (PLGA) microspheres (MS) in nanofibrous scaffolds (NFS) have been shown to control the release of rhPDGF-BB in vitro. In order to investigate the effects of rhPDGF-BB release from MS in NFS on gene expression and enhancement of soft tissue engineering, rhPDGF-BB was incorporated into differing molecular weight (MW) polymeric MS. By controlling the MW of the MS over a range of 6.5 KDa-64 KDa, release rates of PDGF can be regulated over periods of weeks to months in vitro. The NFS-MS scaffolds were divided into multiple groups based on MS release characteristics and PDGF concentration ranging from 2.5-25.0 microg and evaluated in vivo in a soft tissue wound repair model in the dorsa of rats. At 3, 7, 14 and 21 days post-implantation, the scaffold implants were harvested followed by assessments of cell penetration, vasculogenesis and tissue neogenesis. Gene expression profiles using cDNA microarrays were performed on the PDGF-releasing NFS. The percentage of tissue invasion into MS-containing NFS at 7 days was higher in the PDGF groups when compared to controls. Blood vessel number in the HMW groups containing either 2.5 or 25 microg PDGF was increased above those of other groups at 7d (p<0.01). Results from cDNA array showed that PDGF strongly enhanced in vivo gene expression of the CXC chemokine family members such as CXCL1, CXCL2 and CXCL5. Thus, sustained release of rhPDGF-BB, controlled by slow-releasing MS associated with the NFS delivery system, enhanced cell migration and angiogenesis in vivo, and may be related to an induced expression of chemokine-related genes. This approach offers a technology to accurately control growth factor release to promote soft tissue engineering in vivo.

OBJECTIVE

To clarify the role of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway in platelet-derived growth factor (PDGF) induced proliferation in activated pancreatic stellate cells (PSCs).

METHODS

PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. STAT-specific binding activity was assessed by electrophoretic mobility shift assay. Activation of Src, JAK2, STAT1, STAT3, and ERK was determined by Western blotting using anti-phospho-specific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine.

RESULTS

PDGF-BB induced STAT-specific binding activity, and activation of Src, JAK2, STAT1, STAT3, and ERK. Ethanol and acetaldehyde at clinically relevant concentrations decreased basal activation of JAK2 and STAT3. PDGF-induced activation of STAT1 and STAT3 was inhibited by a Src inhibitor PP1 and a JAK2 inhibitor AG490, whereas PDGF-induced activation of ERK was inhibited by PP1, and not by AG490. PDGF-induced proliferation was inhibited by PP1 and AG490 as well as by STAT3 antisense oligonucleotide.

CONCLUSIONS

PDGF-BB activated JAK2-STAT pathway via Src-dependent mechanism. Activation of JAK2-STAT3 pathway, in addition to ERK, may play a role in PDGF-induced proliferation of PSCs.

In the mammalian skeletal system, osteogenesis and angiogenesis are intimately linked during bone growth and regeneration in bone modeling and during bone homeostasis in bone remodeling. Recent studies have expanded our knowledge about the molecular and cellular mechanisms responsible for coupling angiogenesis and bone formation. Type H vessels, termed such because of high expression of Endomucin (Emcn) and CD31, have recently been identified and have the ability to induce bone formation. Factors including platelet-derived growth factor type BB (PDGF-BB), slit guidance ligand 3 (SLIT3), hypoxia-inducible factor 1-alpha (HIF-1α), Notch, and vascular endothelial growth factor (VEGF) are involved in the coupling of angiogenesis and osteogenesis. This review summarizes the current understanding of signaling pathways that regulate type H vessels and how type H vessels modulate osteogenesis. Further studies dissecting the regulation and function of type H vessels will provide new insights into the role of bone vasculature in the metabolism of the skeleton. We also discuss considerations for therapeutic approaches targeting type H vessels to promote fracture healing, prevent pathological bone loss, osteonecrosis, osteoarthritis, and bone metastases.

Obesity is closely associated with the progression of vascular disorders, including atherosclerosis and postangioplasty restenosis. C1q/TNF-related protein (CTRP) 9 is an adipocytokine that is down-regulated in obese mice. Here we investigated whether CTRP9 modulates neointimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation in vivo and in vitro. Left femoral arteries of wild-type (WT) mice were injured by a steel wire. An adenoviral vector expressing CTRP9 (Ad-CTRP9) or β-galactosidase as a control was intravenously injected into WT mice 3 d before vascular injury. Delivery of Ad-CTRP9 significantly attenuated the neointimal thickening and the number of bromodeoxyuridine-positive proliferating cells in the injured arteries compared with that of control. Treatment of VSMCs with CTRP9 protein attenuated the proliferative and chemotactic activities induced by growth factors including platelet-derived growth factor (PDGF)-BB, and suppressed PDGF-BB-stimulated phosphorylation of ERK. CTRP9 treatment dose-dependently increased cAMP levels in VSMCs. Blockade of cAMP-PKA pathway reversed the inhibitory effect of CTRP9 on DNA synthesis and ERK phosphorylation in response to PDGF-BB. The present data indicate that CTRP9 functions to attenuate neointimal formation following vascular injury through its ability to inhibit VSMC growth via cAMP-dependent mechanism, suggesting that the therapeutic approaches to enhance CTRP9 production could be valuable for prevention of vascular restenosis after angioplasty.

BACKGROUND

Cytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period.

METHODS

Fluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample.

RESULTS

Using the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7.

CONCLUSIONS

The current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.

BACKGROUND

There is no information on the effects of the breed, gender and age on the cellular content and growth factor (GF) release from equine pure-platelet rich plasma (P-PRP) and pure-platelet rich gel (P-PRG). The objectives of this study were: 1) to compare the cellular composition of P-PRP with whole blood and platelet poor plasma (PPP); 2) to compare the concentration of transforming GF beta 1 (TGF-β1) and platelet derived GF isoform BB (PDGF-BB) between P-PRP treated with non-ionic detergent (P-PRP+NID), P-PRG (activated with calcium gluconate -CG-), PPP+NID, PPP gel (PPG), and plasma and; 3) to evaluate and to correlate the effect of the breed, gender and age on the cellular and GF concentration for each blood component. Forty adult horses, 20 Argentinean Creole Horses (ACH) and, 20 Colombian Creole Horses (CCH) were included. Data were analyzed by parametric (i.e.: t-test, one way ANOVA) and non parametric (Kruskal-Wallis test, Wilcoxon test) tests. Correlation analysis was also performed by using the Spearman and Pearson tests. A p ≤ 0.05 was set as significant for all tests. All the blood components were compared for platelet (PLT), leukocyte (WBC), TGF-β1 and PDGF-BB concentrations. The effect of the breed, gender and age on these variables was analyzed. A P ≤ 0.05 was accepted as significant for all the tests.

RESULTS

PLT counts were 1.8 and 0.6 times higher in P-PRP than in whole blood and PPP, respectively; WBC counts were 0.5 and 0.1 times lower in P-PRP, in comparison with whole blood and PPP, respectively. TGF-β1 and PDGF-BB concentrations were 2.3 and 262 times higher, respectively, in P-PRG than in plasma, and 0.59 and 0.48 times higher, respectively, in P-PRG than in PPG. P-PRG derived from CCH females or young horses presented significantly (P < 0.001) higher PDGF-BB concentrations than P-PRG derived from ACH males or older horses.

CONCLUSIONS

Our results indicated that P-PRP obtained by a manual method was affected by intrinsic factors such as the breed, gender and age. Equine practitioners should be aware that cellular and GF release from P-PRP/P-PRG could change according with the intrinsic variables associated with a patient in particular.

BACKGROUND

Since the V617F mutation in JAK2 may not be the initiating event in myeloprofilerative disorders (MPDs) we compared molecular changes in neutrophils from patients with polycythemia vera (PV) and essential thrombocythosis (ET), to neutrophils stimulated by G-CSF administration and to normal unstimulated neutrophils

METHODS

A gene expression oligonucleotide microarray with more than 35,000 probes and a microRNA (miR) expression array with 827 probes were used to assess neutrophils from 6 MPD patients; 4 with PV and 2 with ET, 5 healthy subjects and 6 healthy subjects given G-CSF. In addition, neutrophil antigen expression was analyzed by flow cytometry and 64 serum protein levels were analyzed by ELISA.

RESULTS

Gene expression profiles of neutrophils from the MPD patients were similar but distinct from those of healthy subjects, either unstimulated or G-CSF-mobilized. The differentially expressed genes in MPD neutrophils were more likely to be in pathways involved with inflammation while those of G-CSF-mobilized neutrophils were more likely to belong to metabolic pathways. In MPD neutrophils the expression of CCR1 was increased and that of several NF-kappaB pathway genes were decreased. MicroRNA miR-133a and miR-1 in MPD neutrophils were down-regulated the most. Levels of 11 serum proteins were increased in MPD patients including MMP-10, MMP-13, VCAM, P-selectin, PDGF-BB and a CCR1 ligand, MIP-1alpha.

CONCLUSIONS

These studies showed differential expression of genes particularly involved in inflammatory pathways including the NF-kappaB pathway and down-regulation of miR-133a and miR-1. These two microRNAs have been previous associated with certain cancers as well as the regulation of hyperthrophy of cardiac and skeletal muscle cells. These changes may contribute to the clinical manifestations of the MPDs.

BACKGROUND

Vascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc.

METHODS

To demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-β and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression.

RESULTS

We showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-β and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-β was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression.

CONCLUSIONS

BM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth.

The effect of recombinant platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB and -AB) on mitogenesis of human cultured airway smooth muscle (ASM) was examined using the MTT-reduction assay and [3H]thymidine incorporation. Results were correlated with expression of PDGF receptor (PDGFR)-alpha and -beta subunits in the absence and presence of fetal calf serum (FCS). When FCS was absent PDGF-AB and -BB were potent mitogens, whereas PDGF-AA was weakly mitogenic, evoking <20% of the maximum response induced by the B-chain isoforms. When FCS (2.5%) was present, all PDGF isoforms stimulated marked ASM proliferation with similar efficacy and potency. Cross-competition binding analysis in FCS-deprived cells revealed that ASM cells in culture express mainly PDGFR-beta. Preincubation with PDGF-AA or PDGFR-alpha neutralizing antiserum abolished PDGF-AA binding and decreased total receptor number by approximately 15%. The ratio of PDGFR-alpha to beta subunits was approximately 1:8, supported by intense immunofluorescence staining for PDGFR-beta and weak staining for PDGFR-alpha. In parallel studies, uptake of [3H]thymidine stimulated by PDGF-AA, but not PDGF-AB or -BB, was inhibited by PDGFR-alpha immobilization. Western immunoblot analysis confirmed expression of mature PDGFR-alpha and -beta subunits in ASM cells. FCS did not cause any detectable increase in PDGFR-alpha expression or in PDGF-AA binding. These data support a role for PDGFR-beta mediating ASM mitogenesis during FCS-free conditions, but in the presence of FCS, both PDGFR-alpha and -beta subunits are linked to mitogenesis. The enhanced mitogenicity of PDGF-AA in the presence of FCS was independent of any detectable upregulation of PDGFR-alpha, suggesting that the inability of PDGF-AA to promote mitogenesis in the absence of FCS is not simply due to relative numbers of PDGFR-alpha and PDGFR-beta.

Previous evidence has shown that platelet-derived growth factor-BB (PDGF-BB) and fibroblast growth factor-2 (FGF-2) directly interact with high affinity, leading to potent reciprocal inhibitory effects on bovine endothelial cells and rat vascular smooth muscle cells. In this study, we report that PDGF-BB inhibits a series of FGF-2-induced events, such as proliferation of human umbilical vein endothelial cells (HUVECs), FGF-2 cellular internalization, phosphorylation of intracellular signaling factors including p38, rac1/cdc42, MKK4, and MKK3/6, and phosphorylation of FGF-receptor 1 (FGF-R1). PDGF-receptor-alpha (PDGF-Ralpha) was found to mediate PDGF-BB inhibitory effects because its neutralization fully restored FGF-2 mitogenic activity and internalization. Additional biochemical analyses, coimmunoprecipitation experiments, and FRET analysis showed that FGF-R1 and PDGF-Ralpha directly interact in vitro and in vivo and that this interaction is somehow increased in the presence of the corresponding ligands FGF-2 and PDGF-BB. These results suggest that FGF-R1/PDGF-Ralpha heterodimerization may represent a novel endogenous mechanism to modulate the action of these receptors and their ligands and to control endothelial cell function.

Platelet-derived growth factor-BB (PDGF) has been reported to provide tropic support for neurons in the central nervous system. The protective role of PDGF on dopaminergic neurons, especially in the context of HIV-associated dementia (HAD), however, remains largely unknown. Here, we show that exogenous PDGF was neuroprotective against toxicity induced by HIV-1 Tat in primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in PDGF-mediated neuroprotection. TRPC channels are Ca(2+)-permeable, nonselective cation channels with a variety of physiological functions. Blocking TRPC channels with either a blocker or short-interfering RNAs (specific for TRPC 5 and 6) in primary neurons resulted in suppression of both PDGF-mediated neuroprotection as well as elevations in intracellular Ca(2+). PDGF-mediated neuroprotection involved parallel but distinct ERK/CREB and PI3K/Akt pathways. TRPC channel blocking also resulted in suppression of PDGF-induced Pyk2/ERK/CREB activation, but not Akt activation. Relevance of these findings in vivo was further corroborated by intrastriatal injections of PDGF and HIV-1 Tat in mice. Administration of PDGF was able to rescue the dopaminergic neurons in the substantia nigra from Tat-induced neurotoxicity. This effect was attenuated by pre-treatment of mice with the TRP blocker, thus underscoring the novel role of TRPC channels in the neuroprotection mediated by PDGF.

The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor-competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.

Platelet-derived growth factor (PDGF), a key mitogen for liver fat-storing cells (FSC), is a dimeric molecule that occurs as homodimers or heterodimers of related polypeptide chains (PDGF-BB, -AB, and -AA). In chronic inflammation of the liver lobule, any of the three dimeric forms of PDGF derived from multiple sources could potentially interact with FSC. We explored the effects of the three different PDGF isoforms on DNA synthesis and early signal transduction pathways potentially related to PDGF mitogenicity in rat liver FSC. PDGF-BB homodimer and -AB heterodimer induced a marked increase in DNA synthesis, whereas the effect of PDGF-AA homodimer was considerably lower. Moreover, the mitogenicity of each isoform proportionally correlated with their effects on phosphoinositide turnover and intracellular Ca2+. Both the PDGF-BB and -AB dimers likely interact with the PDGF-beta-receptor, although PDGF-AB requires at least one alpha-receptor. The low responsiveness to PDGF-AA could not be accounted for by downregulation of the PDGF-alpha-receptor because FSC expressed very low levels of PDGF-A- and B-chain mRNAs and did not secrete detectable amounts of PDGF activity in the conditioned media. In addition, preincubation of FSC with suramin, a potent inhibitor of PDGF binding to its receptor, failed to increase PDGF-AA-induced DNA synthesis. These results are consistent with a predominant expression of PDGF-beta-receptor in liver FSC, that is linked to phospholipase C activation.