Platelet-derived Growth factor-C Research Articles

Relationships of peripheral IGF-1, VEGF and BDNF levels to exercise-related changes in memory, hippocampal perfusion and volumes in older adults

Animal models point towards a key role of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in mediating exercise-induced structural and functional changes in the hippocampus. Recently, also platelet derived growth factor-C (PDGF-C) has been shown to promote blood vessel growth and neuronal survival. Moreover, reductions of these neurotrophic and angiogenic factors in old age have been related to hippocampal atrophy, decreased vascularization and cognitive decline. In a 3-month aerobic exercise study, forty healthy older humans (60 to 77years) were pseudo-randomly assigned to either an aerobic exercise group (indoor treadmill, n=21) or to a control group (indoor progressive-muscle relaxation/stretching, n=19). As reported recently, we found evidence for fitness-related perfusion changes of the aged human hippocampus that were closely linked to changes in episodic memory function. Here, we test whether peripheral levels of BDNF, IGF-I, VEGF or PDGF-C are related to changes in hippocampal blood flow, volume and memory performance. Growth factor levels were not significantly affected by exercise, and their changes were not related to changes in fitness or perfusion. However, changes in IGF-I levels were positively correlated with hippocampal volume changes (derived by manual volumetry and voxel-based morphometry) and late verbal recall performance, a relationship that seemed to be independent of fitness, perfusion or their changes over time. These preliminary findings link IGF-I levels to hippocampal volume changes and putatively hippocampus-dependent memory changes that seem to occur over time independently of exercise. We discuss methodological shortcomings of our study and potential differences in the temporal dynamics of how IGF-1, VEGF and BDNF may be affected by exercise and to what extent these differences may have led to the negative findings reported here.

Clinical significance of platelet derived growth factor-C and -D in gastric cancer.

Platelet-derived growth factor (PDGF)-C and PDGF-D are frequently upregulated in human cancers and play important roles in tumor progression, angiogenesis and metastasis. However, the distribution, frequency and prognostic value of PDGF-C and PDGF-D expression in gastric cancer have not been clarified. The present study evaluated the association between expression of PDGF-C and PDGF-D, clinicopathological factors and outcomes, in patients with gastric cancer. Gastric adenocarcinoma tumor samples were obtained from 204 patients who underwent curative gastrectomy between 2003 and 2007. The expression of PDGF-C and PDGF-D was analyzed by immunohistochemical staining. High expression of PDGF-C and PDGF-D was detected in 114 (56%) and 151 (74%) tumors, respectively. PDGF-D expression was significantly associated with tumor depth (P=0.039), histopathology (P<0.01), tumor stage (P=0.01) and recurrence (P<0.01), whereas PDGF-C expression correlated only with histopathology (P=0.05). High PDGF-D expression was also associated with significantly shorter relapse-free survival (RFS) time (P<0.01), whilst high PDGF-C expression was associated with marginally, but not significantly, shorter RFS (P=0.10). On multivariate analysis, high PDGF-D expression was determined to be an independent prognostic factor (hazard ratio, 3.3; 95% confidence interval, 1.20–9.4; P=0.02). These findings indicate that high PDGF-D expression is strongly associated with tumor progression, recurrence, distant metastasis and poor outcomes in patients with gastric cancer. PDGF-D may therefore be an independent prognostic factor and a novel therapeutic target.

Inhibition of microRNA‐214 ameliorates hepatic fibrosis and tumor incidence in platelet‐derived growth factor C transgenic mice

Differentially regulated microRNA (miRNA) are associated with hepatic fibrosis; however, their potential usefulness for blocking hepatic fibrosis has not been exploited fully. We examined the expression of miRNA in the liver of a transgenic mouse model in which platelet-derived growth factor C (PDGF-C) is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis and steatosis and the eventual development of hepatocellular carcinoma (HCC). Robust induction of miR-214 correlated with fibrogenesis in the liver of Pdgf-c Tg mice, atherogenic high-fat diet-induced NASH mice, and patients with chronic hepatitis B or C. Pdgf-c Tg mice were injected with locked nucleic acid (LNA)-antimiR-214 via the tail vein using Invivofectamine 2.0 and the degree of hepatic fibrosis and tumor incidence were evaluated. Pdgf-c Tg mice treated with LNA-antimiR-214 showed a marked reduction in fibrosis and tumor incidence compared with saline or LNA-miR-control-injected control mice. In vitro, LNA-antimiR-214 significantly ameliorated TGF-β1-induced pro-fibrotic gene expression in Lx-2 cells. MiR-214 targets a negative regulator of EGFR signaling, Mig-6. Mimic-miR-214 decreased the expression of Mig-6 and increased the levels of EGF-mediated p-EGFR (Y1173 and Y845) and p-Met (Tyr1234/1235) in Huh-7 cells. Conversely, LNA-antimiR-214 repressed the expression of these genes. In conclusion, miR-214 appears to participate in the development of hepatic fibrosis by modulating the EGFR and TGF-β signaling pathways. LNA-antimiR-214 is a potential therapy for the prevention of hepatic fibrosis.

PROTEIN KINASE C-α INHIBITS ANGIOGENESIS INDUCED BY PLATELET-DERIVED GROWTH FACTOR C IN HYPERGLYCEMIC ENDOTHELIAL CELLS

Diabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that expression of platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) was downregulated in ischemic limbs of diabetic mice, although the

Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells.

BackgroundDiabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) were downregulated in ischemic limbs of diabetic mice, although the underlying mechanisms remained elusive. Protein kinase C (PKC) is a family of serine/threonine kinases and is known to be involved in angiogenesis. The purpose of this study is to elucidate the mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes.MethodsHuman umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) cultured in normoglycemic or hyperglycemic conditions were examined. We also examined the effects of PKC inhibition on the PDGF-C/PDGFR-α axis in endothelial cells exposed to hyperglycemia.ResultsHyperglycemia inhibited proliferation and decreased viability of both HUVECs and HMVECs. Hyperglycemic endothelial cells exhibited decreased PDGFR-α expression both at messenger RNA (mRNA) and protein levels, while there was no significant change in expression of PDGF-C. We also found that expression of PKC-α, one of the PKC isoforms, was increased in hyperglycemic endothelial cells and that inhibition of PKC upregulated PDGFR-α expression in these cells. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by PDGF-C was significantly attenuated in hyperglycemic endothelial cells, whereas inhibition of PKC effectively reversed these inhibitory effects. Moreover, inhibition of PKC also promoted angiogenesis induced by PDGF-C in hyperglycemic endothelial cells, which was not observed in vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis.ConclusionsThese findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of hyperglycemia through upregulation of PKC. Targeting PKC to restore PDGF-C signaling might be a novel therapeutic strategy for the treatment of vascular complications in diabetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0180-9) contains supplementary material, which is available to authorized users.

The Association of Serum IL-33 and sST2 with Breast Cancer.

Breast cancer is one of the most common malignant diseases in women. The main cause of death from breast cancer is its metastases at distant sites in the body. Interleukin-33 (IL-33) is a cytokine of the IL-1 family and found overexpressed in various cancers. The aim of the present study was to explore the association of serum IL-33 and sST2 with breast cancer. Here, the serum levels of Interleukin-33 (IL-33) and sST2 were found significantly higher in breast cancer patients than in healthy volunteers. Serum levels of vascular endothelial growth factor (VEGF), metalloproteinase-11 (MMP-11), and platelet-derived growth factor-C (PDGF-C) were also greater in breast cancer patients compared to healthy volunteers. We found that serum levels of IL-33 or sST2 were positively correlated with the serum levels of VEGF, MMP-11, and PDGF-C. Moreover, breast cancer dataset downloaded from The Cancer Genome Atlas showed that patients with higher level of MMP-11 or PDGF-C expression had shorter survival time than those with lower level of these proteins. In conclusion, IL-33 and sST2 may serve as noninvasive diagnosis markers for breast cancer. IL-33 and sST2 were significantly associated with MMP-11 or PDGF-C which indicated poor prognosis of breast cancer patients.

Immunohistochemical distribution of platelet-derived growth factor-C and platelet-derived growth factor receptor-α in small intestine of rats treated with capsaicin

The purpose of this study was to examine the effect of capsaicin administration immunohistochemically on platelet-derived growth factor (PDGF)-C and platelet-derived growth factor receptor (PDGFR)-\alpha release in the small intestine tissues of rats. Rats were divided into three groups: group 1 (subcutaneous drug administration), group 2 (oral drug administration), and group 3 (subcutaneous sham administration). Histometric measurements and counts were performed in order to determine whether there was a difference between groups in terms of height of villi, depth of crypts, and goblet cell count in small intestine. The small intestine was examined by Crossman?s triple staining method and streptavidin-biotin peroxidase complex method. As a result of statistical analysis, it was found that body weight increased in group 3, while it decreased in groups 1 and 2. Capsaicin administration increased height of villi, depth of crypts, and count of goblet cells located on villi and crypts. Immunohistochemical examinations revealed that capsaicin administration increased PDGF-C and PDGFR-? release. It was concluded that capsaicin has a stimulatory effect on growth and is an enhancer for PDGF-C and PDGFR-? release.

Immunohistochemical examination of effects of kefir, koumiss and commercial probiotic capsules on platelet derived growth factor-c and platelet derived growth factor receptor-alpha expression in mouse liver and kidney

We investigated using immunohistochemistry the effects of kefir, koumiss and commercial probiotic capsules on the expression of platelet derived growth factor-c (PDGF-C) and platelet derived growth factor receptor-alpha (PDGFR-α) in mouse liver and kidney. Mice were assigned to four groups: group 1 was given commercial probiotic capsules, group 2 was given kefir, group 3 was given koumiss and group 4 was untreated. After oral administration for 15 days, body weights were recorded and liver and kidney tissue samples were obtained. Hematoxylin and eosin staining was used to examine histology. PDGF-C and PDGFR-α in liver and kidney were localized using the streptavidin-biotin peroxidase complex method (ABC). We found that the weights of the mice in the kefir, koumiss and commercial probiotic capsules groups increased compared to the control group. No differences in liver and kidney histology were observed in any of the experimental groups. Kefir, koumiss and the commercial probiotic preparation increased PDGF-C and PDGFR-α expression.

Post-transcriptional up-regulation of PDGF-C by HuR in advanced and stressed breast cancer.

Breast cancer is a heterogeneous disease characterized by multiple genetic alterations leading to the activation of growth factor signaling pathways that promote cell proliferation. Platelet-derived growth factor-C (PDGF-C) is overexpressed in various malignancies; however, the involvement of PDGF-C in breast cancers and the mechanisms underlying PDGF-C deregulation remain unclear. Here, we show that PDGF-C is overexpressed in clinical breast cancers and correlates with poor prognosis. PDGF-C up-regulation was mediated by the human embryonic lethal abnormal vision-like protein HuR, which stabilizes the PDGF-C transcript by binding to two predicted AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). HuR is up-regulated in hydrogen peroxide-treated or ultraviolet-irradiated breast cancer cells. Clinically, HuR levels are correlated with PDGF-C expression and histological grade or pathological tumor-node-metastasis (pTNM) stage. Our findings reveal a novel mechanism underlying HuR-mediated breast cancer progression, and suggest that HuR and PDGF-C are potential molecular candidates for targeted therapy of breast cancers.

“Special research presentation” endothelin regulation of cyr61 in uterine leiomyomas

Genetic studies have identified imbalances in angiogenic factors such as cysteine –rich protein 61 (Cyr61) and platelet derived growth factor-C (PDGFC) in women with uterine leiomyomas. Low oxygen conditions, hypoxia, have been shown as a possible mechanism to increase endothelin-1 and potentially lead to aberrant expression of Cyr61 and PDGFC. The objective of this study was to determine if hypoxia induces Cyr61 secretion from fibroid smooth muscle cells (mSMCs) to levels comparable to secretion from myometrial smooth muscle cells (fSMCs) through activation of the Endothelin-A (ETA) receptor.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Clinical Significance of Platelet-Derived Growth Factor-C Expression in Colorectal Cancer

Platelet-derived growth factors (PDGFs) are known to be associated with tumor growth and angiogenesis through their activation of the receptor tyrosine kinases, PDGF receptors alpha and beta. Several studies revealed the participation of the PDGF family in colorectal cancer (CRC). However, the role of platelet derived growth factor-C (PDGFC) in CRC is less well studied. This study aimed to determine the correlation between PDGFC expression and the prognosis of patients with CRCs. Tumor samples were obtained from patients with CRC who underwent surgical resection between 2002 and 2006. The mRNA expression of PDGFC was investigated by quantitative reverse transcription-polymerase chain reaction in 85 patients with stage I-IV CRC. PDGFC protein expression was analyzed by immunohistochemistry, and the relationship between PDGFC protein expression and clinicopathologic features was investigated in 245 patients with stage I-III CRC. PDGFC mRNA expression in cancer tissues was significantly higher in patients with distant metastases than in those without metastases (P = 0.016). PDGFC protein overexpression was associated with significantly worse overall and relapse-free survival (P P - 6.081, P

Tissue-type plasminogen activator is not necessary for platelet-derived growth factor-c activation

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.

Paracrine activation of hepatic stellate cells in platelet‐derived growth factor C transgenic mice: Evidence for stromal induction of hepatocellular carcinoma

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor-stromal interactions in the liver.

Bilaterally cleft lip and bilateral thumb polydactyly with triphalangeal component in a patient with two De novo deletions of HSA 4q32 and 4q34 involving PDGFC, GRIA2, and FBXO8 genes

We report on a newborn boy with a bilateral cleft of the primary palate, duplicated triphalangeal thumbs, and a patent foramen ovale. During childhood he had moderate developmental delay. Brain MRI at 4 years was normal. The concurrence of non-syndromic clefts of the lip/palate (CL/P) and duplicated thumbs with triphalangeal component has, to our knowledge, not been reported so far. In our case, array-CGH analysis documented two de novo deletions (∼1.2 Mb and ∼400 Kb) of the long arm of chromosome 4, containing four genes: platelet-derived growth factor C (PDGFC), glycine receptor beta subunit (GLRB), glutamate receptor ionotropic AMPA2 (GRIA2), and F-box protein 8 gene (FBXO8). PDGFC codes for a mesenchymal cell growth factor already known to be associated with clefts of the lip. Pdgfc(-/-) mice have skeletal anomalies, and facial schisis resembling human cleft/lip palate. GRIA2 codes for a ligand-activated cation channel that mediates the fast component of postsynaptic excitatory currents in neurons, and may be linked to cognitive dysfunction. FBXO8, a gene of unknown function, is a member of the F-box gene family, among which FBXW4, within the minimal duplicated region associated with human split-hand/foot malformation type 3 (SHFM type 3). The presence of overlapping deletions in patients who do not share the same phenotype of our case suggests incomplete penetrance, and a possible effect of modifier genetic factors.

Platelet-derived growth factor C promotes revascularization in ischemic limbs of diabetic mice

Platelet-derived growth factor C (PDGF-C) has been reported to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis in vivo remains poorly understood. VEGF has been employed as a major molecular tool to induce therapeutic angiogenesis. However, VEGF therapy is not very effective in models of cardiovascular diseases associated with diabetes, and the mechanisms of this phenomenon still remain to be elucidated. We used a murine model of hind limb ischemia and of streptozotocin-induced diabetes. Expression of PDGF-C and its receptor PDGFR-α were markedly upregulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as a VEGF-neutralizing antibody. Mice deficient in PDGF-C exhibited reduced blood flow recovery after ischemia compared with wild-type mice, confirming a strong proangiogenic activity of PDGF-C. Next, we injected an expression vector encoding PDGF-C into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the groups treated with PDGF-C compared with controls. Attenuation of angiogenic responses to ischemia has been reported in patients with diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. We tested this hypothesis by inducing diabetes by intraperitoneal injection of streptozotocin. Expression levels of PDGF-C at baseline and after ischemia were significantly lower in limb tissues of diabetic mice than in those of control mice, whereas expression levels of other members of the PDGF family and VEGF were not changed or were even higher in diabetic mice. Introduction of VEGF complementary DNA expression plasmid vector into ischemic limbs did not improve blood flow recovery. However, these changes were effectively reversed by additional introduction of the PDGF-C complementary DNA plasmid vector. These results indicate that downregulation of PDGF-C expression in limb tissues of diabetic mice contributes to impaired angiogenesis and suggest that introduction of PDGF-C might be a novel strategy for therapeutic angiogenesis, especially in the diabetic state. Angiogenesis and arteriogenesis after ischemia are attenuated in most diabetic patients, although the precise mechanisms remain unclear. Platelet-derived growth factors (PDGFs) have a variety of functions on many cell types, and PDGF-C stimulates angiogenesis and revascularizes ischemic tissues. This study indicates the role for PDGF-C as a critical regulator of impaired angiogenesis of diabetes and suggests that PDGF-C might be a novel target for the treatment of ischemic cardiovascular diseases in diabetes.

Circulating angiogenic factors as predictors of benefit from bevacizumab (bev) beyond progression in metastatic colorectal cancer (mCRC): Traslational analyses from the phase III BEBYP trial.

3603 Background: TML and BEBYP trials demonstrated that the strategy of prosecuting bev beyond progression is effective in mCRC. Previous analyses from phase II studies showed that a modulation of plasma angiogenic factors occurs during 1st-line treatment with chemotherapy (CT) + bev and a wide variability in soluble Vascular Endothelial Growth Factor Receptor-2 (sVEGFR-2) levels was observed at the time of progression. Moving from preliminary analyses in murine models we selected a pool of candidate ligands to be tested in the clinical setting. Methods: sVEGFR2, Placental Growth Factor (PlGF), Platelet-Derived Growth Factor-C, basic Fibroblast Growth Factor, Angiopoietin-2 and soluble Tie-2 were assessed by ELISA on plasma samples collected at baseline in a cohort of 59 patients enrolled in phase III BEBYP trial of 2nd-line CT ± bev beyond progression to a bev-containing first-line regimen. Plasma levels were defined high or low adopting the median values as cut-off. Results: A significant interaction between treatment arm and baseline sVEGFR-2 levels was observed (p=0.036). Among 30 patients with high sVEGFR-2 levels, the prosecution of bev was associated with a significant benefit in terms of PFS (median: 10.4 vs 3.4 months, HR 0.37 [95%CI 0.10-0.58], p=0.0015), that was not evident among 29 patients with low sVEGFR-2 levels (5.4 vs 5.0 months, HR 0.98 [95%CI 0.45-2.11], p=0.956). Despite a trend towards a greater benefit from bev among 30 patients with high PlGF levels (HR 0.45 [95%CI 0.13-0.86]), no interaction between treatment arm and baseline PlGF levels was observed (p=0.210). Combined analysis of sVEGFR-2 and PlGF showed that prosecuting bev provided a substantial benefit in PFS in the subgroup with high levels of both ligands (10.5 vs 2.3 months, HR 0.25 [95%CI 0.01-0.45], p=0.043). Conclusions: sVEGFR-2 levels at the time of first progression may predict benefit from prosecuting bev. Interesting results from simultaneous analysis of sVEGFR-2 and PlGF may be affected by a pronounced subgrouping and should be considered cautiously. Given their potential clinical value, these data need prospective confirmation. Clinical trial information: NCT00720512.

Circulating angiogenic factors as predictors of benefit from bevacizumab (bev) beyond progression in metastatic colorectal cancer (mCRC): Translational analyses from the phase III BEBYP trial.

382 Background: TML and BEBYP trials demonstrated that the strategy of prosecuting bev beyond progression is effective in mCRC. Previous analyses from phase II studies showed that a dynamic modulation of plasma angiogenic factors occurs during first-line treatment with chemotherapy (CT) plus bev and a wide variability in soluble Vascular Endothelial Growth Factor Receptor 2 (sVEGFR2) levels was observed at the time of progression. Moving from preliminary analyses in murine models we selected a pool of candidate ligands to be tested in the clinical setting. Methods: sVEGFR2, Placental Growth Factor (PlGF), Platelet-Derived Growth Factor-C, basic Fibroblast Growth Factor, Angiopoietin-2 and sTie-2 were assessed by ELISA on plasma samples collected at baseline in a cohort of 59 patients enrolled in phase III BEBYP trial of 2nd-line CT ± bev beyond progression to a bev-containing first-line regimen. Plasma levels were defined high or low adopting the median as cut-off. Results: A significant interaction between treatment arm and baseline sVEGFR2 levels was observed (p=0.036). Among 30 patients with high sVEGFR2 levels, the prosecution of bev was associated with a significant benefit in terms of PFS (median: 10.4 vs 3.4 months, HR 0.37 [95%CI:0.10-0.58], p=0.0015), that was not evident among 29 patients with low sVEGFR2 levels (5.4 vs 5.0 months, HR 0.98 [95%CI:0.45-2.11], p=0.956). Despite a trend towards a greater benefit from bev among 30 patients with high PlGF levels (HR 0.45 [95%CI: 0.13-0.86]), no interaction between treatment arm and baseline PlGF levels was observed (p=0.210). Combined analysis of sVEGFR2 and PlGF showed that prosecuting bev provided a substantial benefit in PFS in the subgroup with high levels of both ligands (10.5 vs 2.3 months, HR 0.25 [95%CI:0.01-0.45], p=0.043). Conclusions: sVEGFR2 levels at the time of first progression may predict benefit from the prosecution of bev. Impressive results deriving from concomitant testing of sVEGFR2 and PlGF may be affected by excessive subgrouping and should be considered cautiously. Given their potential clinical value these data need prospective confirmation. Clinical trial information: NCT00720512.

Construction of a plasmid for expression of rat platelet-derived growth factor C and its effects on proliferation, migration and adhesion of endothelial progenitor cells

Endothelial progenitor cells (EPCs) play a key role in restoring endothelial function and enhancing angiogenesis. Platelet-derived growth factor C (PDGF-C) is a newly discovered member of the PDGF family that binds to the PDGFR-α homodimer and the PDGFR-α/β heterodimer. Currently, the biological effects of PDGF-C on EPCs proliferation, migration and adhesion are not well understood. In this study, the full-length coding sequence (CDS) region for the PDGF-C gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified PDGF-C product was digested and inserted into the pMD 19-T simple vector and then subcloned into a pIRES2-EGFP plasmid to construct the pIRES2-EGFP-PDGF-C eukaryotic expression vector. After it was transfected to EPCs, the expression of PDGF-C protein in EPCs was verified by Western blotting analysis. Finally, we investigated the effects of PDGF-C protein overexpression on EPCs proliferation, migration and adhesion. In conclusion, we constructed a recombinant eukaryotic expression vector containing the complete CDS region of PDGF-C and expressed the full-length and functional PDGF-C protein successfully. Furthermore, PDGF-C promoted EPCs proliferation, migration and adhesion. This offers promise for the development of new therapeutic strategies for improving neovascularization and repair of blood vessel endothelium in patients with ischemic heart disease or peripheral arterial occlusive disease.

Expression of platelet-derived growth factor-C and insulin-like growth factor I in hepatic stellate cells is inhibited by miR-29

MicroRNAs are short noncoding, endogenous RNA species that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of mRNAs. Recently, it was shown that miR-29 inhibits expression of extracellular matrix proteins such as collagens, suggesting an antifibrotic function of miR-29. In the present study, we now investigated the role of miR-29 in profibrogenic growth factor expression as a further central mechanism of fibrosis. Screening of databases revealed putative miR-29 target sequences in the mRNA of platelet-derived growth factor (PDGF)-B, PDGF-B receptor, PDGF-C, vascular endothelial growth factor-A, and insulin-like growth factor (IGF)-I. To analyze miR-29 interaction with the predicted binding sites, we cloned the 3′-UTR sequences of the putative targets in fusion to the luciferase-reporter coding sequence. Functional miR-29 binding to PDGF-C and IGF-I mRNA sequences, but not to the corresponding mutants, was then proven by reporter assays. Hepatic stellate cells (HSC) that transdifferentiate into myofibroblasts, producing extracellular matrix proteins and profibrogenic growth factors, for example, the members of the PDGF family, are crucial for liver fibrosis. Myofibroblastic transition of primary HSC resulted in the loss of miR-29, but in a significant increase of PDGF-C and IGF-I. Compensation of reduced miR-29 levels by miR-29 overexpression in myofibroblastic HSC was followed by a definitive repression of IGF-I and PDGF-C synthesis. After experimental fibrosis, induced by bile-duct occlusion, miR-29 expression was shown to be reduced, but IGF-I and PDGF-C expression was upregulated, correlating inversely to the miR-29 pattern. Thus, we conclude that miR-29, downregulated during fibrosis, acts as an antifibrogenic mediator not only by targeting collagen biosynthesis, but also by interfering with profibrogenic cell communication via PDGF-C and IGF-I.