The impairment of ILK related angiogenesis involved in cardiac maladaptation after infarction

Background

Integrin linked kinase (ILK), as an important component of mechanical stretch sensor, can initiate cellular signaling response in the heart when cardiac preload increases. Previous work demonstrated increased ILK expression could induce angiogenesis to improved heart function after MI. However the patholo-physiological role of ILK in cardiac remodeling after MI is not clear.

Method and Results

Hearts were induced to cardiac remodeling by infarction and studied in Sprague-Dawley rats. Until 4 weeks after infarction, ILK expression was increased in non-ischemic tissue in parallel with myocytes hypertrophy and compensatory cardiac function. 8 weeks later, when decompensation of heart function occurred, ILK level returned to baseline. Followed ILK alternation, vascular endothelial growth factor (VEGF) expression and phosphorylation of endothelial nitric oxide synthase (eNOS) was significantly decreased 8 weeks after MI. Histology study also showed significantly microvessel decreased and myocytes loss 8 weeks paralleled with ILK down-regualtion. While ILK expression was maintained by gene delivery, tissue angiogenesis and cardiac function was preserved during cardiac remodeling.

Conclusion

Temporally up-regulation of ILK level in non-ischemic myocytes by increased external load is associated with beneficial angiogenesis to maintain infarction-induced cardiac hypertrophy. When ILK expression returns to normal, this cardiac adaptive response for infarction is weaken. Understanding the ILK related mechanism of cardiac maladaptation leads to a new strategy for treatment of heart failure after infarction.     

In-vivo visualization of tumor microvessel density and response to anti-angiogenic treatment by high resolution MRI in mice

Purpose

Inhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance.

Experimental Design

We established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice.

Results

Using microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size.

Conclusions

We present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects has high potential in applications to preclinical and clinical trials.     

Increased expression of integrin-linked kinase improves cardiac function and decreases mortality in dilated cardiomyopathy model of rats

Aims

Integrin-linked kinase (ILK) is a multifunctional kinase linking the extracellular matrix to intracellular signaling pathways, whose activation in the heart gives rise to a number of functional consequences. The aim of this study is to demonstrate the therapeutic and survival benefit of cardiac ILK overexpression in a rat model of dilated cardiomyopathy.

Methods and Results

The dilated cardiomyopathy model was generated in rats by intraperitoneal administration of six equal doses of doxorubicin over a 2 week period. Five weeks after the first injection, echocardiographic analysis demonstrated impaired cardiac function and, at that point, recombinant adenoviral vector harboring ILK cDNA or vehicle was injected into the myocardium, and the rats re-studied 4 weeks later. Compared with vehicle injection, ILK treatment ameliorated inflammatory cell infiltration and cardiomyocyte degeneration, as well as left ventricular dilation and dysfunction. ILK treatment was also associated with a reduction in apoptosis and an increase in proliferation of cardiomyocytes, as well as decreased oxidative stress and autophagic vacuole accumulation. Importantly, mortality was lower in rats following ILK treatment than in those following vehicle injection. In cultured neonatal rat cardiomyocytes, we also found that ILK overexpression protected against doxorubicin-induced apoptosis, giving rise to an increase in their proliferation.

Conclusions

These data demonstrate for the first time that ILK gene therapy improves cardiac function and survival in a model of dilated cardiomyopathy, and this may be mediated through suppression of inflammation, prevention of ventricular remodeling, inhibition of cardiomyocyte apoptosis and autophagy, and stimulation of cardiomyocyte proliferation.     

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

Background

Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

Methodology/Principal Findings

Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

Conclusions/Significance

Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.     

In Silico Design and Biological Evaluation of a Dual Specificity Kinase Inhibitor Targeting Cell Cycle Progression and Angiogenesis

Background

Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.

Methodology

We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.

Conclusions

We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.     

Plexin-B1 activates NF-κB and IL-8 to promote a pro-angiogenic response in endothelial cells

Background

The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.

Methods/Principal Findings

Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.

Conclusion/Significance

These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.     

Tetrahydrohyperforin and Octahydrohyperforin Are Two New Potent Inhibitors of Angiogenesis

Background

We have previously shown that hyperforin, a phloroglucinol derivative found in St. John''s wort, behaves as a potent anti-angiogenic compound. To identify the reactive group(s) mainly involved in this anti-angiogenic effect, we have investigated the anti-angiogenic properties of a series of stable derivatives obtained by oxidative modification of the natural product. In addition, in the present work we have studied the role of the four carbonyl groups present in hyperforin by investigating the potential of some other chemically stable derivatives.

Methodology/Principal Findings

The experimental procedures included the analysis of the effects of treatment of endothelial cells with these compounds in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Our study with hyperforin and eight derivatives shows that the enolized β-dicarbonyl system contained in the structure of hyperforin has a dominant role in its antiangiogenic activity. On the other hand, two of the tested hyperforin derivatives, namely, tetrahydrohyperforin and octahydrohyperforin, behave as potent inhibitors of angiogenesis. Additional characterization of these compounds included a cell specificity study of their effects on cell growth, as well as the in vivo Matrigel plug assay.

Conclusions/Significance

These observations could be useful for the rational design and chemical synthesis of more effective hyperforin derivatives as anti-angiogenic drugs. Altogether, the results indicate that octahydrohyperforin is a more specific and slightly more potent antiangiogenic compound than hyperforin.     

Overexpression of EFEMP1 Correlates with Tumor Progression and Poor Prognosis in Human Ovarian Carcinoma

Objective

This study was to explore the role of EFEMP1 in ovarian tumor progression and its relationship with prognosis of ovarian carcinoma.

Methods

EFEMP1 mRNA and protein expressions in normal ovarian tissue, ovarian tumor, high invasive subclones and low invasive subclones were evaluated by immunohistochemistry and real time RT-PCR. Serum EFEMP1 levels in patients with ovarian tumor were measured by ELISA assay. To assess the angiogenic properties of EFEMP1, VEGF and tumor microvessel density were analyzed in ovarian carcinoma by immunohistochemistry.

Results

EFEMP1 expression was up-regulated in ovarian carcinoma, positively correlated with MVD and VEGF, and its overexpression and high serum levels were significantly associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. EFEMP1 expression was also found to be over-expressed in the highly invasive subclones compared with the low invasive subclones.

Conclusion

EFEMP1 is a newly identified gene over-expressed in ovarian cancer, associated with poor clinicopathologic features and promotes angiogenesis. This study shows that EFEMP1 may serve as a new prognostic factor and a therapeutic target for patients with ovarian cancer in the future.     

Prostate specific membrane antigen (PSMA) regulates angiogenesis independently of VEGF during ocular neovascularization

Background

Aberrant growth of blood vessels in the eye forms the basis of many incapacitating diseases and currently the majority of patients respond to anti-angiogenic therapies based on blocking the principal angiogenic growth factor, vascular endothelial growth factor (VEGF). While highly successful, new therapeutic targets are critical for the increasing number of individuals susceptible to retina-related pathologies in our increasingly aging population. Prostate specific membrane antigen (PSMA) is a cell surface peptidase that is absent on normal tissue vasculature but is highly expressed on the neovasculature of most solid tumors, where we have previously shown to regulate angiogenic endothelial cell invasion. Because pathologic angiogenic responses are often triggered by distinct signals, we sought to determine if PSMA also contributes to the pathologic angiogenesis provoked by hypoxia of the retina, which underlies many debilitating retinopathies.

Methodology/Principal Findings

Using a mouse model of oxygen-induced retinopathy, we found that while developmental angiogenesis is normal in PSMA null mice, hypoxic challenge resulted in decreased retinal vascular pathology when compared to wild type mice as assessed by avascular area and numbers of vascular tufts/glomeruli. The vessels formed in the PSMA null mice were more organized and highly perfused, suggesting a more ‘normal’ phenotype. Importantly, the decrease in angiogenesis was not due to an impaired hypoxic response as levels of pro-angiogenic factors are comparable; indicating that PSMA regulation of angiogenesis is independent of VEGF. Furthermore, both systemic and intravitreal administration of a PSMA inhibitor in wild type mice undergoing OIR mimicked the PSMA null phenotype resulting in improved retinal vasculature.

Conclusions/Significance

Our data indicate that PSMA plays a VEGF-independent role in retinal angiogenesis and that the lack of or inhibition of PSMA may represent a novel therapeutic strategy for treatment of angiogenesis-based ocular diseases.     

LF-15 & T7, Synthetic Peptides Derived from Tumstatin,Attenuate Aspects of Airway Remodelling in a Murine Model of Chronic OVA-Induced Allergic Airway Disease

Background

Tumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its anti-angiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the αVβ3 integrin.

Methods

Primary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR) was then examined using a murine model of chronic OVA-induced allergic airways disease.

Results

The LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters.

Conclusion

The LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo.     

Anti-angiogenic and anti-inflammatory properties of kahweol, a coffee diterpene

Background

Epidemiological studies have shown that unfiltered coffee consumption is associated with a low incidence of cancer. This study aims to identify the effects of kahweol, an antioxidant diterpene contained in unfiltered coffee, on angiogenesis and key inflammatory molecules.

Methodology/Principal Findings

The experimental procedures included in vivo angiogenesis assays (both the chicken and quail choriallantoic membrane assay and the angiogenesis assay with fluorescent zebrafish), the ex vivo mouse aortic ring assay and the in vitro analysis of the effects of treatment of human endothelial cells with kahweol in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Additionally, two inflammation markers were determined, namely, the expression levels of cyclooxygenase 2 and the levels of secreted monocyte chemoattractant protein-1. We show for the first time that kahweol is an anti-angiogenic compound with inhibitory effects in two in vivo and one ex vivo angiogenesis models, with effects on specific steps of the angiogenic process: endothelial cell proliferation, migration, invasion and tube formation on Matrigel. We also demonstrate the inhibitory effect of kahweol on the endothelial cell potential to remodel extracellular matrix by targeting two key molecules involved in the process, MMP-2 and uPA. Finally, the anti-inflammatory potential of this compound is demonstrated by its inhibition of both COX-2 expression and MCP-1 secretion in endothelial cells.

Conclusion/Significance

Taken together, our data indicate that, indeed, kahweol behaves as an anti-inflammatory and anti-angiogenic compound with potential use in antitumoral therapies. These data may contribute to the explanation of the reported antitumoral effects of kahweol, including the recent epidemiological meta-analysis showing that drinking coffee could decrease the risk of certain cancers.     

Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic,anti-inflammatory and anti-catabolic properties

Introduction

Chondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1β) is regulated by CS, employing a novel quantitative proteomic approach.

Methods

Human articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1β 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results.

Results

We could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1β, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1.

Conclusion

We have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1β. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.     

Combination Therapy of VEGF-Trap and Gemcitabine Results in Improved Anti-Tumor Efficacy in a Mouse Lung Cancer Model

Background

Angiogenesis is essential for the growth and metastasis of cancer. Although anti-angiogenic agents, particularly vascular endothelial growth factor (VEGF) inhibitors, have exhibited single-agent activity, there is considerable interest in combining these novel drugs with conventional chemotherapy reagents to achieve an optimal clinical efficacy. The objective of this study was to evaluate the benefits of the combination therapy of vascular endothelial growth factor trap (VEGF-Trap) with gemcitabine in a lung tumor model.

Methods

A luciferase-expressing Lewis lung carcinoma (LLC) model was established in C57BL/6J mice and tumor-bearing mice were randomized into control, VEGF-Trap, gemcitabine and VEGF-Trap/gemcitabine combination groups. Tumor growth and animal survival were monitored. Tumor microvessel density and cell proliferation were evaluated by CD31 and Ki-67 immunohistochemical analysis. TUNEL assay was performed to detect apoptotic cells. The protein levels of Cyclin D1, Pro-Caspase-3, Bcl-2, MMP2 and MMP9 in tumor extracts were examined by western blot.

Results

VEGF-Trap in combination with gemcitabine showed significantly enhanced inhibition of tumor growth and prolonged mouse survival compared to the VEGF-Trap or gemcitabine monotherapy. The VEGF-Trap/gemcitabine combination therapy not only potently inhibited tumor angiogenesis and cell proliferation, but also increased cellular apoptosis within tumor tissues. In addition, the combination treatment markedly down-regulated the expression of proliferation, anti-apoptosis and invasion related proteins.

Conclusion

Combination therapy using VEGF-Trap and gemcitabine resulted in improved anti-tumor efficacy in a lung cancer model and VEGF-Trap/gemcitabine combination might represent a promising strategy in the treatment of human lung cancer.     

Quantitative Susceptibility Mapping Differentiates between Blood Depositions and Calcifications in Patients with Glioblastoma

Objectives

The application of susceptibility weighted imaging (SWI) in brain tumor imaging is mainly used to assess tumor-related “susceptibility based signals” (SBS). The origin of SBS in glioblastoma is still unknown, potentially representing calcifications or blood depositions. Reliable differentiation between both entities may be important to evaluate treatment response and to identify glioblastoma with oligodendroglial components that are supposed to present calcifications. Since calcifications and blood deposits are difficult to differentiate using conventional MRI, we investigated whether a new post-processing approach, quantitative susceptibility mapping (QSM), is able to distinguish between both entities reliably.

Materials and Methods

SWI, FLAIR, and T1-w images were acquired from 46 patients with glioblastoma (14 newly diagnosed, 24 treated with radiochemotherapy, 8 treated with radiochemotherapy and additional anti-angiogenic medication). Susceptibility maps were calculated from SWI data. All glioblastoma were evaluated for the appearance of hypointense or hyperintense correlates of SBS on the susceptibility maps.

Results

43 of 46 glioblastoma presented only hyperintense intratumoral SBS on susceptibility maps, indicating blood deposits. Additional hypointense correlates of tumor-related SBS on susceptibility maps, indicating calcification, were identified in 2 patients being treated with radiochemotherapy and in one patient being treated with additional anti-angiogenic medication. Histopathologic reports revealed an oligodendroglial component in one patient that presented calcifications on susceptibility maps.

Conclusions

QSM provides a quantitative, local MRI contrast, which reliably differentiates between blood deposits and calcifications. Thus, quantitative susceptibility mapping appears promising to identify rare variants of glioblastoma with oligodendroglial components non-invasively and may allow monitoring the role of calcification in the context of different therapy regimes.     

Anti-Angiogenic Effects of a Mutant Endostatin: A New Prospect for Treating Retinal and Choroidal Neovascularization

Purpose

Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).

Methods

M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.

Results

The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.

Conclusion

The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.     

miRNome traits analysis on endothelial lineage cells discloses biomarker potential circulating microRNAs which affect progenitor activities

Background

Endothelial progenitor cells (EPCs) play a fundamental role in not only blood vessel development but also post-natal vascular repair. Currently EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Both EPC types assist angiogenesis and have been linked to ischemia-related disorders, including coronary artery disease (CAD).

Results

We found late EPCs are more mobile than early EPCs and matured endothelial cells (ECs). To pinpoint the mechanism, microRNA profiles of early EPCs late EPCs, and ECs were deciphered by small RNA sequencing. Obtained signatures made up of both novel and known microRNAs, in which anti-angiogenic microRNAs such as miR-221 and miR-222 are more abundant in matured ECs than in late EPCs. Overexpression of miR-221 and miR-222 resulted in the reduction of genes involved in hypoxia response, metabolism, TGF-beta signalling, and cell motion. Not only hamper late EPC activities in vitro, both microRNAs (especially miR-222) also hindered in vivo vasculogenesis in a zebrafish model. Reporter assays showed that miR-222, but not miR-221, targets the angiogenic factor ETS1. In contrast, PIK3R1 is the target of miR-221, but not miR-222 in late EPCs. Clinically, both miR-221-PIK3R1 and miR-222-ETS1 pairs are deregulated in late EPCs of CAD patients.

Conclusions

Our results illustrate EPCs and ECs exploit unique miRNA modalities to regulate angiogenic features, and explain why late EPC levels and activities are reduced in CAD patients. These data will further help to develop new plasma biomarkers and therapeutic approaches for ischemia-related diseases or tumor angiogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-802) contains supplementary material, which is available to authorized users.     

Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Background

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.

Methodology/Principal Findings

Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC₂₀). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K_m for MnATP is ∼150-fold less than that for MgATP).

Conclusions/Significance

Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases.     

Transcriptional analysis of VEGF-D and TGFβ genes in MCF7 cells exposed to saponin isolated from Holothuria leucospilota (sea cucumber)

Background:

Marine natural products contain a wide range of bioactive compounds with therapeutic properties that have revealed crucial properties in the treatment of some diseases. Some of these compounds have recently received considerable attention for drug discovery. In this study we examined the anti-angiogenic effect of saponin isolated from Holothuria leucospilota (sea cucumber) through evaluation of vascular endothelial growth factor D (VEGF-D) and transforming growth factor-β (TGFβ) expression in a breast cancer cell line.

Methods:

To investigate the effect of SCS on VEGF-D and TGF-β expression in breast cancer cells, the cells were treated with various concentrations of sample. After 48 h the viability of the cells was evaluated by trypan blue staining, and VEGF-D and TGFβ mRNA expression was were evaluated by real time-PCR.

Results:

Our results revealed that SCS can suppress cell viability and VEGF-D and TGFβ mRNA expression in breast cancer cells. Sea cucumber saponin at a concentration of 12 μg/ml inhibited VEGF-D and TGFβ expression more than 90% compared with controls.

Conclusion:

Findings suggest that SCS could inhibit tumor growth via inhibition of angiogenesis.Key Words: Sea cucumber, Saponin, Angiogenesis, Anticancer     

Integrin-linked kinase overexpression and its oncogenic role in promoting tumorigenicity of hepatocellular carcinoma

Background

Integrin-linked kinase (ILK) was first discovered as an integrin β1-subunit binding protein. It localizes at the focal adhesions and is involved in cytoskeleton remodeling. ILK overexpression and its dysregulated signaling cascades have been reported in many human cancers. Aberrant expression of ILK influenced a wide range of signaling pathways and cellular functions. Although ILK has been well characterized in many malignancies, its role in hepatocellular carcinoma (HCC) is still largely unknown.

Methodology/Principal Findings

Quantitative PCR analysis was used to examine ILK mRNA expression in HCC clinical samples. It was shown that ILK was overexpressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumorous livers. The overall ILK expression level was significantly higher in tumorous tissues (P = 0.004), with a significant stepwise increase in expression level along tumor progression from tumor stage I to IV (P = 0.045). ILK knockdown stable clones were established in two HCC cell lines, BEL7402 and HLE, and were subjected to different functional assays. Knockdown of ILK significantly suppressed HCC cell growth, motility and invasion in vitro and inhibited tumorigenicity in vivo. Western blot analysis revealed a reduced phosphorylated-Akt (pAkt) at Serine-473 expression in ILK knockdown stable clones when compared to control clones.

Conclusion/Significance

This study provides evidence about the clinical relevance of ILK in hepatocarcinogenesis. ILK was found to be progressively elevated along HCC progression. Here our findings also provide the first validation about the oncogenic capacity of ILK in vivo by suppressing its expression in HCC cells. The oncogenic role of ILK is implicated to be mediated by Akt pathway.