Activation of β-platelet-derived growth factor receptor (β-PDGFR) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification, which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor hypoxia-inducible factor-1α (HIF-1α). Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D-expressing cells. Treatment of vector control or PDGF-B-expressing cells with the HIF-1α activator CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis, and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D-transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.

Chinese dragon's blood, the red resin of Dracaena cochinchinensis, one of the famous traditional medicines, has been used to promote blood circulation, disperse blood stasis, stop bleeding, relieve pain and muscle regeneration for thousands of years. The aims of this study were to evaluate the anti-atherosclerotic effect of Longxuetongluo Capsule (LTC), which made by total phenolic compounds of Chinese dragon's blood, in high cholesterol diet (HCD)-induced atherosclerosis model rats and explore the possible mechanism. Atherosclerosis rats were induced by administration of HCD for 4 weeks and treated with atorvastatin (2.08mg/kg/d) or various concentrations of LTC (81, 162 and 324mg/kg/d) for additional 4 weeks. Body weight (BW), lipid profiles, serum VCAM-1, ICAM-1, MCP-1, AST and ALT were then tested. Histopathological evaluation of aorta and liver were determined by hematoxylin and eosin staining. NF-κB expression in aorta was detected by Immunohistochemical staining. Meanwhile, the inhibition effects of LTC on the migration and proliferation and Intracellular Ca2+ levels induced by PDGF-BB were also evaluated in rat aortic smooth muscle cells (A7r5). The results demonstrated that LTC produced a significant anti-atherosclerotic activity in terms of reduction in serum lipids and lipoprotein profile, VCAM-1, ICAM-1, MCP-1, AST, ALT levels, and increase in HDL-c level compared to atherosclerotic group. Rats treated with LTC not only attenuated the pathological region and atheroma formation, but also reduced hepatic steatosis and inflammatory cell infiltration. Immunohistochemical analysis showed LTC reduced NF-κB expression in aorta. Furthermore, PDGF-BB induced proliferation and migration of A7r5 and intracellular calcium rise were also abrogated by LTC. The results indicate that LTC prevents atherosclerosis and fatty liver by controlling lipid metabolism, the underlying mechanism may attributed to its anti-inflammation activity, regulation of the vascular smooth muscle function and intracellular calcium signaling.

PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine and endocrine mechanisms. We investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells.

Keywords: D. melanogaster; genetics; genomics.

According to the model of "response to injury," the arterial endothelium is occasionally injured in hyperlipidemia, hypertension, diabetes mellitus and in other states known as risk factors. The ensuing inflammatory response is modulated by cytokines and growth factors, among them platelet-derived growth factor (PDGF), and monocyte chemoattractant protein-1 (MCP-1). In two independent studies, we demonstrated that mRNA levels for PDGF-A and -B and for MCP-1 are reduced after ingestion of n-3 fatty acids by human volunteers. This reduction persists after monocyte stimulation/differentiation by adherence. Moreover, the reduction is brought about only by dietary n-3 fatty acids and not by other classes of unsaturated fatty acids (n-6 or n-9). This appears to be one major mechanism of action of reduced progression/increased regression of established coronary artery disease by ingestion of 1.5 g/d n-3 fatty acids, as assessed by coronary angiography in a randomized placebo-controlled double-blind intervention study in 223 patients. The study was conducted according to "Good Clinical Practice," comprehensive rules regulating investigations with pharmaceutical compounds. Together, our investigations lend support to the importance of PDGF-A, PDGF-B, and MCP-1 in the pathogenesis of atherosclerosis, and the beneficial role of n-3 fatty acids therein.

The involvement of growth factors (GFs) in the pathogenesis of lumbar intervertebral disc (ID) herniation and the spontaneous resorption of herniated ID fragments remains only partially elucidated. A simultaneous assessment of the transcript levels of numerous GFs and their association with clinical and epidemiological profiles of human ID herniation would provide valuable insight into the biology and clinical course of the disease. In the present study, we examined simultaneously the transcript levels of vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF‑β1), basic fibroblast growth factor 2 (bFGF2), platelet derived growth factor (PDGF) isoforms and receptors, epidermal growth factor (EGF) and insulin growth factor‑1 (IGF‑1) in herniated and control ID specimens and investigated their correlation with the clinicopathological profiles of patients suffering from symptomatic lumbar ID herniation. GF mRNA expression levels were determined by RT-qPCR in 63 surgical specimens from lumbar herniated discs and 10 control ID specimens. Multiple positive correlations were observed between the transcript levels of the GFs examined in the ID herniation group. VEGF mRNA expression was significantly increased in the protruding compared with the extruded discs. Intense and acute pain significantly upregulated the PDGF transcript levels. Significant negative correlations were observed between the patient body mass index and the transcript levels of VEGF and PDGF receptors. Our findings support the hypothesis of the involvement of GFs in the natural history of ID herniation. GFs synergistically act in herniated IDs. Increased VEGF expression possibly induces the neovascularization process in the earliest stages of ID herniation. PDGF‑C and ‑D play a role in the acute phase of radiculopathy in a metabolic response for tissue healing. A molecular effect, in addition to the biomechanical effect of obesity in the pathogenesis of ID herniation is also implied.

BACKGROUND

To develop a protocol for obtaining autologous platelet rich plasma in healthy individuals and to determine the concentration of five major growth factors before platelet activation. This protocol could be integrated into the guidelines of good clinical practice and research in regenerative medicine.

METHODS

Platelet rich plasma was isolated by centrifugation from 38 healthy men and 42 women ranging from 18 to 59 years old. The platelet count and quantification of growth factors were analyzed in eighty samples, stratified for age and gender of the donor. Analyses were performed using parametric the t-test or Pearson's analysis for non-parametric distribution. P < 0.05 was considered statistically significant.

RESULTS

Our centrifugation protocol allowed us to concentrate basal platelet counts from 1.6 to 4.9 times (mean = 2.8). There was no correlation between platelet concentration and the level of the following growth factors: VEGF-D (r = 0.009, p = 0.4105), VEGF-A (r = 0.0068, p = 0.953), PDGF subunit AA (p = 0.3618; r = 0.1047), PDGF-BB (p = 0.5936; r = 0.6095). In the same way, there was no correlation between donor gender and growth factor concentrations. Only TGF-β concentration was correlated to platelet concentration (r = 0.3163, p = 0.0175).

CONCLUSIONS

The procedure used allowed us to make preparations rich in platelets, low in leukocytes and red blood cells, and sterile. Our results showed biological variations in content of growth factors in PRP. The factors influencing these results should be further studied.

Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin DD promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.

Intussusceptive angiogenesis (IA) is currently considered an important alternative and complementary form of sprouting angiogenesis (SA). Conversely, intussusceptive lymphangiogenesis (IL) is in an initial phase of study. We compare their morphofunctional characteristics, since many can be shared by both processes. To that end, the following aspects are considered: A) The concept of IA and IL as the mechanism by which blood and lymphatic vessels split, expand and remodel through transluminal pillar formations (hallmarks of intussusception). B) Terminology and historical background, with particular reference to the group of Burri, including Djonov and Patan, who initiated and developed the vessel intussusceptive concept in blood vessels. C) Incidence in normal (e.g. in the sinuses of developing lymph nodes) and pathologic conditions, above all in vessel diseases, such as dilated veins in hemorrhoidal disease, intravascular papillary endothelial hyperplasia (IPEH), sinusoidal hemangioma, lobular capillary hemangioma, lymphangiomas/lymphatic malformations and vascular transformation of lymph nodes. D) Differences and complementarity between vessel sprouting and intussusception. E) Characteristics of the cover (endothelial cells) and core (connective tissue components) of pillars and requirements for pillar identification. F) Structures involved in pillar formation, including endothelial contacts of opposite vessel walls, interendothelial bridges, merged adjacent capillaries, vessel loops and spilt pillars. G) Structures resulting from pillars with intussusceptive microvascular growth, arborization, remodeling and segmentation (compartmentalization). H) Influence of intussusception in the morphogenesis of vessel tumors/ pseudotumors; and I) Hemodynamic and molecular control of vessel intussusception, including VEGF, PDGF BB, Hypoxia, Notch, Endoglobin and Nitric oxide.

Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblast-like cells from these lung biopsies, which stained for alpha-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and post-translational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiation, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin DPDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function.

Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRalpha and beta proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRbeta and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRbeta expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRalpha and PDGF-B mRNA expression. We suggest that inhibition of PDGFRbeta expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB.

The migration and proliferation of vascular smooth muscle cells (SMC) into the neointima are important early events in the development of atherosclerosis and post-angioplasty restenosis. The stimulation of SMC migration by platelet derived growth factor (PDGF) involves the induction of protein kinase C activity. Using immunoblot techniques, we demonstrated that rat aortic SMC express a pattern of PKC isoforms which includes PKC-alpha, delta, epsilon, zeta and eta. Upon exposure to PDGF-BB, a fraction of PKC-delta was rapidly translocated from the cytosol to the post-nuclear particulate fraction at 15 seconds and reached an apparent maximum at 30 minutes. In contrast, PKC-alpha and zeta were not translocated by PDGF-BB, TGF-beta 1, which inhibits PDGF-induced DNA synthesis and chemotaxis, reduced the immunoreactive levels of PKC-delta and blocked the PDGF-induced translocation of PKC-delta to the particulate fraction. This suggests that the activation of PKC-delta by translocation to the particulate fraction may play an important role in the control of vascular smooth muscle cell migration by PDGF and TGF-beta 1.

BACKGROUND

Network inference of gene expression data is an important challenge in systems biology. Novel algorithms may provide more detailed gene regulatory networks (GRN) for complex, chronic inflammatory diseases such as rheumatoid arthritis (RA), in which activated synovial fibroblasts (SFBs) play a major role. Since the detailed mechanisms underlying this activation are still unclear, simultaneous investigation of multi-stimuli activation of SFBs offers the possibility to elucidate the regulatory effects of multiple mediators and to gain new insights into disease pathogenesis.

METHODS

A GRN was therefore inferred from RA-SFBs treated with 4 different stimuli (IL-1 β, TNF- α, TGF- β, and PDGF-D). Data from time series microarray experiments (0, 1, 2, 4, 12 h; Affymetrix HG-U133 Plus 2.0) were batch-corrected applying 'ComBat', analyzed for differentially expressed genes over time with 'Limma', and used for the inference of a robust GRN with NetGenerator V2.0, a heuristic ordinary differential equation-based method with soft integration of prior knowledge.

RESULTS

Using all genes differentially expressed over time in RA-SFBs for any stimulus, and selecting the genes belonging to the most significant gene ontology (GO) term, i.e., 'cartilage development', a dynamic, robust, moderately complex multi-stimuli GRN was generated with 24 genes and 57 edges in total, 31 of which were gene-to-gene edges. Prior literature-based knowledge derived from Pathway Studio or manual searches was reflected in the final network by 25/57 confirmed edges (44%). The model contained known network motifs crucial for dynamic cellular behavior, e.g., cross-talk among pathways, positive feed-back loops, and positive feed-forward motifs (including suppression of the transcriptional repressor OSR2 by all 4 stimuli.

CONCLUSIONS

A multi-stimuli GRN highly concordant with literature data was successfully generated by network inference from the gene expression of stimulated RA-SFBs. The GRN showed high reliability, since 10 predicted edges were independently validated by literature findings post network inference. The selected GO term 'cartilage development' contained a number of differentiation markers, growth factors, and transcription factors with potential relevance for RA. Finally, the model provided new insight into the response of RA-SFBs to multiple stimuli implicated in the pathogenesis of RA, in particular to the 'novel' potent growth factor PDGF-D.

This study aimed to investigate whether platelet-derived growth factor D (PDGF-D) is a prognostic biomarker and is associated with platinum resistance in epithelial ovarian cancer, which has not been studied by others previously.

In this study, we detected expression of PDGF-D in ovarian cancer tissues through immunohistochemistry and Western blotting. Furthermore, we analyzed the association between PDGF-D expression and clinicopathological features including prognosis in epithelial ovarian cancer. Statistical analyses were performed by using χ test, log-rank test, Cox regression test, and Kaplan-Meier method.

High PDGF-D expression is positively correlated with International Federation of Gynecology and Obstetrics stage (P < 0.001), histologic grade (P < 0.001), lymph node metastasis (P = 0.022), and poor prognosis (P < 0.001). Platelet-derived growth factor D in platinum-resistant cases is overexpressed compared with that in platinum-sensitive cases (P < 0.001). Obstetrics stage (P = 0.029) and PDGF-D overexpression (P < 0.001) are independently correlated with platinum resistance.

Our study indicates that PDGF-D overexpression is an independent predictor of platinum-based chemotherapy resistance and that it may also be a potential biomarker for targeted therapy and poor prognosis.

Platelet-derived growth factor D (PDGF-D) has been shown to mediate cellular processes of importance in cancer progression. This study aimed to investigate the expression and putative involvement of PDGF-D signaling in colorectal carcinogenesis. PDGF-D was expressed in vascular endothelial cells in tumor and normal tissues. PDGF-D stimulation of cells altered genes of importance in carcinogenic processes. In addition, PDGF-D increased the proliferation rate while imatinib inhibited these effects. PDGF-D and its PDGF receptor beta (PDGFR-β) are expressed in colorectal cancer and blockage of PDGF-D/PDGFR-β signaling using tyrosine kinase inhibitors, such as imatinib, might be important in inhibiting tumor-promoting actions.

In the pathogenesis of renal fibrosis, oxidative stress (OS) enhances the production of reactive oxygen species (ROS) leading to sustained cell growth, inflammation, excessive tissue remodelling and accumulation, which results in the development and acceleration of renal damage. In our previous work (Eltoweissy et al., 2011) we established protein DJ-1 (PARK7) as an important ROS scavenger and key player in renal cell response to OS. In the present study we investigated the impact of profibrogenic agonists on DJ-1 and shed light on the role of this protein in renal fibrosis. Treatment of renal fibroblasts and epithelial cells with the profibrogenic agonist ANG II or PDGF resulted in a significant up-regulation of DJ-1 expression parallel to an increase in the expression of fibrosis markers. Monitoring of DJ-1 expression in kidney extract and tissue sections from a renal fibrosis mouse model (Col4a3-deficient) revealed a disease grade dependent regulation of the protein. Overexpression of DJ-1 prompted cell resistance to OS in both fibroblasts and epithelial cells. Furthermore overexpression of DJ-1, involved in ROS scavenging, in which glutamic acid 18 (E18) is mutated to either to aspartic acid (D) or glutamine (Q) resulted in a significant increase in cell death under OS in the case of E18D mutation, whereas E18Q mutation did not impact significantly the cell response to OS, revealing the importance of the acidic group for the ROS scavenging activity of the DJ-1 protein more than the nature of the amino acid itself. Affinity precipitation of interaction partners of DJ-1 and its mutants revealed an important role of annexin A1 and A5 in the mechanism of action of DJ-1 in anti-oxidative stress response.

Malabaricone C (Mal-C), isolated from nutmeg, is known to exert a variety of pharmacological activities. However, the effect of Mal-C on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of Mal-C on proliferation and migration of primary rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Treatment of RASMCs with Mal-C induced both protein and mRNA expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Mal-C-mediated HO-1 induction was inhibited by treatment with actinomycin D or by cycloheximide. SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor), and N-acetylcysteine (NAC, an antioxidant) did not suppress Mal-C-induced HO-1 expression. In contrast, LY294002 (a PI3K inhibitor) blocked Mal-C-induced HO-1 expression. Moreover, RASMCs treated with Mal-C exhibited activation of AKT in a dose- and time-dependent manner. Treatment of RASMCs with Mal-C increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), which is a key regulator of HO-1 expression, and this translocation was also inhibited by LY294002. Consistent with the notion that HO-1 has protective effects against VSMCs, Mal-C remarkably inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of RASMCs. However, inhibition of HO-1 significantly attenuated the inhibitory effects of Mal-C on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that Mal-C could suppress PDGF-induced proliferation and migration of RASMCs through Nrf2 activation and subsequent HO-1 induction via the PI3K/AKT pathway, and may be a potential HO-1 inducer for preventing or treating vascular diseases.

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10(-7) M, 6.3 × 10(-9) M, and 4.4 × 10(-7) M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.

OBJECTIVE

To determine the quantitative and qualitative composition of growth factors (PDGF-AA, PDGF-BB, VEGF, VEGF-D, FGF-acid, FGF-basic) and platelets in various modifications of APRP.

METHODS

Blood of 12 male volunteers (control group) and 12 patients with ED was used to prepare APRP and the subsequently determine the concentration of growth factors. The growth factor concentrations (FGF acid, FGF basic, PDGF-AA, PDGF-BB, VEGF, VEGF-D) was determined using a flow cytometry-based xMAP Luminex (Gen-Probe) system.

RESULTS

Concentration of platelets in APRP obtained by two stage centrifugation, reached 1480 (1120-1644) in the control group and 1232 (956-1502) in patients with ED. The concentration of growth factors in the samples prepared without preliminary freezing was: PDGF-AA 842 (22-3700), PDGF-BB 2837 (1460-4100), FGF-basic 7.9 (0.28-127), FGF-acid 3, 4 (0.14-11), VEGF 19 (4.6-46), VEGF-D 21 (14-38). After thawing, the concentration of all growth factors in the samples increased.

CONCLUSIONS

The study findings suggest that the mechanism of erectile function recovery following the use of APRP is through the active substances detected in APRP, i.e. FGF-basic, PDGF-AA, PDGF-BB, VEGF, VEGF-D and FGF-acid. Also, the study showed that the content of growth factors in APRP after of freezing/thawing is higher than in APRP that has not been frozen. This is due to the cell membrane destruction at extremely low temperatures during freezing.

Vascular endothelial growth factor D (VEGF-D) is a member of the VEGF/PDGF superfamily that has been implicated in angiogenesis and lymphangiogenesis. We have isolated a chick cDNA that shows homology with VEGF-D (also known as FIGF, c-fos-induced growth factor) of other species. Here, we describe the expression pattern of cVegf-D in chick embryos. In the limb buds, cVegf-D shows a dynamic expression pattern that is restricted to the mesenchyme of the posterior region. cVegf-D expression is also detected in the ectoderm and mesenchyme of the head region, somites, notochord and pharyngeal arches. We also report on the capability of Sonic hedgehog and retinoic acid to regulate cVegf-D expression.

Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5'-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5'-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.

The effects of high (28mM) D-glucose (HG) on the cell cycle progression were investigated in rat aorta vascular smooth muscle cells (VSMCs) in primary culture, using an immunocytochemical analysis of cell-cycle-specific nuclear antigens. HG had no effect on the cell cycle of the serum-deprived G0 cells, whereas platelet-derived growth factor (PDGF) stimulated the entry of the G0 cells to the G1 phase without a further progression to the S and M phases. HG, but neither mannitol nor L-glucose, stimulated the progression of the PDGF-pretreated G1 cells to the S and M phases, which was blocked by calphostin-C, a protein kinase C (PKC) blocker. HG did not affect the cytosolic Ca2+ concentration ([Ca2+]i). These data suggest that HG has no competent effect on the G0 cells and acts as a progression growth factor (to stimulate the cell cycle from the G1 to the S/M) in VSMCs through the mechanism, which may be insensitive to [Ca2+]i and mediated by PKC.