The expression of AGGF1, FOXC2, and E-cadherin in esophageal carcinoma and their clinical significance.

Angiogenic factor with G-patch and FHA domain 1 (AGGF1) is a newly initiator of angiogenesis. Forkhead box C2 (FOXC2) that is a member of the winged spiral transcription factor family plays an important role in epithelial-mesenchymal transition (EMT). Epithelial-cadherin (E-cad) that is an adhesion molecule is also involved in EMT. The purpose of this study is to investigate the expression of AGGF1, FOXC2, and E-cad in esophageal squamous cell carcinoma (ESCC) and their clinical significance.Immunohistochemistry was performed to investigate the expression of AGGF1, FOXC2, and E-cad in 170 ESCC specimens and corresponding normal esophageal mucosa tissues. Follow-up data was also collected.The positive rates of AGGF1 and FOXC2 expression were significantly higher in ESCC group when compared with the control group; the positive rate of E-cad expression was significantly lower in ESCC group when compared with the control group. Positive rates of AGGF1, FOXC2, and E-cad expression were significantly associated with grades of differentiation, tumor grades, lymph node metastasis stages, as well as tumor-node-metastasis stages. Kaplan-Meier analysis demonstrated that positive expression of AGGF1 or FOXC2 for ESCC patients had significantly unfavorably overall survival time when compared with patients with negative expression of AGGF1 or FOXC2; and positive expression of E-cad for ESCC patients had significantly longer overall survival time when compared with patients with negative expression of E-cad. Multivariate analysis indicated that AGGF1, FOXC2, and E-cad expression and tumor-node-metastasis stages were postoperative independent prognostic factors for ESCC patients.AGGF1, FOXC2, and E-cad may be considered promising biomarkers of ESCC patients' prognosis.

Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage.

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.

Activity of the human immortalized endothelial progenitor cell line HEPC-CB.1 supporting in vitro angiogenesis.

The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells' potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine.

Angiogenic protein synthesis after photobiomodulation therapy on SHED: a preliminary study.

This study evaluated the viability, proliferation, and protein expression after photobiomodulation (PBM) of stem cell from human exfoliated deciduous teeth (SHED). The groups were the following: G1 (2.5 J/cm2), G2 (3.7 J/cm2), and control (not irradiated). According to the groups, cells were irradiated with InGaAlP diode laser at 660 nm wavelength, continuous mode, and single time application. After 6 h, 12 h, and 24 h from irradiation, the cell viability and proliferation, and the protein expression were analyzed by MTT, crystal violet, and ELISA multiplex assay, respectively. Twenty-four hours after PBM, SHED showed better proliferation. Over time in the supernatant, all groups had an increase at the levels of VEGF-C, VEGF-A, and PLGF. In the lysate, the control and G2 exhibited a decrease of the VEGF-A, PECAM-1, and PLGF expression, while control and G3 decreased VEGF-C, VEGF-A, and PDGF expression. The dosimetries of 2.5 J/cm2 and 3.7 J/cm2 maintained viability, improved proliferation, and synthesis of the angiogenic proteins in the supernatant in the studied periods on SHED.

Significance of Angiogenic Growth Factors in Bovine Ocular Squamous Cell Carcinoma.

Bovine ocular squamous cell carcinoma (BOSCC) is the most common and economically significant neoplasm of the eye in cattle. This study investigated the role of angiogenic growth factors in the pathogenesis of BOSCC. Eighteen cases of BOSCC were classified histopathologically according to the degree of differentiation. Normal upper and lower eyelids and third eyelids collected from the right and left eyes of six healthy cattle aged 1-3 years, that had been presented for slaughter to abattoirs, served as controls. Transcription of genes encoding the angiogenic growth factors vascular endothelial growth factor-C (VEGF-C), basic fibroblast growth factor (bFGF), platelet-derived growth factor-C (PDGF-C) and platelet-derived growth factor receptor-α (PDGFR-α) was determined by quantitative real-time polymerase chain reaction (RT-PCR) in tissue obtained from paraffin wax blocks. Immunohistochemistry (IHC) was utilized to detect intensity of expression and tissue distribution of these growth factors. IHC results revealed that bFGF and PDGF-C were elevated significantly (P >0.05) and VEGF-C expression was decreased in BOSCC compared with healthy control tissue. PDGR-α expression was elevated; however, the difference, compared with control tissues, was not significant. RT-PCR results showed an inverse relationship to the results of IHC; where protein levels were elevated their corresponding mRNA levels were decreased or vice-versa. Angiogenic regulators therefore appear to play a role in the pathogenesis of BOSCC.

A Short Bout of Exercise Prior to Stroke Improves Functional Outcomes by Enhancing Angiogenesis.

Stroke remains a significant unmet clinical need with limited therapeutic options. The peculiar feature of ischemic stroke is the interruption in brain circulation, resulting in a cascade of detrimental cerebrovasculature alterations. Treatment strategies designed to maintain potency of the cerebrovasculature may protect against stroke. The present study assessed the effects of short bouts of exercise prior to stroke induction and characterized cerebral blood flow and motor functions in vivo. Adult Sprague-Dawley rats were exposed to a single short bout of exercise (30-min or 60-min forced running wheel) then subjected to transient middle cerebral artery occlusion (MCAO). Non-exercise stroke rats served as controls while non-stroke rats represented shams. Cerebral blood flow (CBF) was evaluated by laser Doppler at baseline (prior to MCAO), during MCAO, and during reperfusion. Behavioral tests using the elevated body swing test was conducted at baseline, day 0 (day of stroke), and at days 1 and 3 after stroke. Animals that received exercise displayed typical alterations in CBF after stroke, but exhibited improved motor performance compared to non-exercise rats. Exercised stroke rats showed a reduction in infarct size and an increased number of surviving cells in the peri-infarct area, with a trend towards prolonged duration of the exercise. Immunofluorescence staining and Western blot analysis of the peri-infarct area revealed increased levels of endothelial markers/angiogenesis markers, VEGF, VEGFR-2, and Ang-2, and endothelial progenitor cell marker CD34+ in exercise groups compared with the controls. These results demonstrated that prophylactic exercise affords neuroprotection possibly by improving cerebrovascular potency.

Hypoxia Inducible Factor Expression and Angiogenesis - Analysis in the Pituitary Gland and Patterns of Death.

BACKGROUND/AIM: We investigated the expression of angiogenesis and hypoxia markers in the adenohypophysis and neurohypophysis of patients who died from various acute or chronic diseases. MATERIALS AND METHODS: Paraffin-embedded material of pituitary glands (97 patients) was investigated immunohistochemically for vascular density (CD31) and the expression of vascular endothelial growth factor (VEGF) and of hypoxia inducible factors HIF1α and HIF2α. RESULTS: Vascular density, and HIF1α/HIF2α reactivity is directly related with VEGF expression in the pituitary gland, suggesting that the HIF pathway may regulate the vascular density and blood flow in the gland under hypoxic conditions. HIF2α appears to be a key regulator in neurohypophysis, whilst in adenohypophysis HIF1α and HIF2α are equally expressed. Chronic conditions, including alcoholism and substance abuse, seem to activate the HIF pathway in both neuro- and adeno-hypophysis. CONCLUSION: The HIF pathway has an important role in regulating vascular density and blood flow in the pituitary gland.

Angiogenic Growth Factors for Coronary Artery Disease: Current Status and Prospects.

Although there have been advances in coronary artery bypass grafting and percutaneous coronary intervention, some patients who have ischemic coronary artery disease (CAD) are ineligible for revascularization due to suboptimal anatomy. Cardiac angiogenesis is not only a physiological response to ischemia or hypoxia but also a potential target of therapeutic strategies. Preclinical studies have shown a great enthusiasm on therapeutic angiogenesis for ischemic CAD. However, the latest trials provided the limited evidence on its efficacy. This article aims to discuss the physiological process of angiogenesis, the characteristic of angiogenic growth factors, delivery system, and clinical and preclinical studies, which can provide a novel insight into the therapeutic angiogenesis for CAD.

The Profile of Angiogenic Factors in Vitreous Humor of the Patients with Proliferative Diabetic Retinopathy.

OBJECTIVE: To explore the expression profile of angiogenic factors associated with proliferative diabetic retinopathy (PDR). METHODS: Undiluted vitreous humor samples were obtained from 10 diabetic patients with PDR (10 eyes) and 9 non-diabtic patients (9 eyes). The concentrations of 60 angiogenic factors in the vitreous humor samples were measured by RayBio Angiogenic Cytokine Antibody Array. Some differentially expressed factors were further confirmed in vitreous humor by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the non-diabetic controls, 20 differentially expressed factors with more than 1.50 fold changes were detected in patients with PDR. The median concentration of vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), angiopoietin (ANG)-1, ANG-2, urokinase-type plasminogen activator receptor (uPAR), Follistatin and matrix metalloproteinases 9 (MMP-9) was significantly increased in vitreous samples from PDR compared to controls (P < 0.05). However, (MCP)-1, Angiogenin and Leptin was significantly lower in PDR eyes compared to controls (P < 0.05). In the verification assay using ELISA, ANG-1, ANG-2, IL-6, VEGF, MMP-9, hepatocyte growth factor (HGF) and placenta growth factor (PIGF) concentration were increased in patients with PDR compared to controls (all P-values < 0.05). CONCLUSION: This is the first report of a comprehensive multiplex analysis to identify angiogenic factors associated with PDR. These angiogenic factors may contribute to the pathogenesis of PDR and may be targets for therapeutic strategies of PDR.

Metabolically conditioned media derived from bone marrow stromal cells or human skin fibroblasts act as effective chemoattractants for mesenchymal stem cells.

BACKGROUND: The main goal of bone tissue engineering has been the generation of healthy bone in order to replace affected tissue. Therefore, optimized biomaterials are needed which allow the survival and growth of mesenchymal stem cells. Until now the key challenge in the clinical application of cell-based tissue engineering bone implants was poor diffusion of oxygen into the tissue, making functional blood vessel networks a necessity. With their ability to evolve into different cell types, to expand extensively in vitro, and to release paracrine soluble factors, bone marrow stromal cells (BMSC) are highly attractive for tissue engineering. During the last years hypoxia became a proven method to control proliferation, differentiation, and pluripotency of BMSC. Here we applied different methods to characterize metabolically conditioned media (MCM) in comparison to hypoxia conditioned media (HCM) and evaluated their ability to attract BMSC in 2-D migration assays. METHODS: BMSC and fibroblasts of human origin were isolated and cultivated to obtain HCM and MCM. Both media were characterized by angiogenesis arrays, cytokine arrays, and ELISA for selected factors. 2-D migration tests were performed with Corning Transwell®-96 permeable support chambers with porous polyester membranes with a pore size of 8.0 µm. RESULTS: Characterization of HCM and MCM revealed that the concentration of angiogenic factors was higher in MCM than in HCM. However, the chemoattractive capacity of MCM for BMSC was equivalent to that of HCM. HCM and MCM produced by human skin fibroblasts attracted human BMSC as efficiently as HCM and MCM produced by human BMSC. CONCLUSIONS: HCM and MCM have a high chemoattractive capacity for BMSC. Both conditioned media harbor high concentrations of angiogenic factors which are important for angiogenesis and cell migration. Both chemoattracting conditioned media can also be derived from skin fibroblasts which can easily be obtained from patients in individualized therapy approaches.

Histone Deacetylase 1 Depletion Activates Human Cardiac Mesenchymal Stromal Cell Proangiogenic Paracrine Signaling Through a Mechanism Requiring Enhanced Basic Fibroblast Growth Factor Synthesis and Secretion.

BACKGROUND: Cardiac mesenchymal cell (CMC) administration improves cardiac function in animal models of heart failure. Although the precise mechanisms remain unclear, transdifferentiation and paracrine signaling are suggested to underlie their cardiac reparative effects. We have shown that histone deacetylase 1 (HDAC1) inhibition enhances CMC cardiomyogenic lineage commitment. Here, we investigated the impact of HDAC1 on CMC cytokine secretion and associated paracrine-mediated activities on endothelial cell function. METHODS AND RESULTS: CMCs were transduced with shRNA constructs targeting HDAC1 (shHDAC1) or nontarget (shNT) control. Cytokine arrays were used to assess the expression of secreted proteins in conditioned medium (CM) from shHDAC1 or shNT-transduced CMCs. In vitro functional assays for cell proliferation, protection from oxidative stress, cell migration, and tube formation were performed on human endothelial cells incubated with CM from the various treatment conditions. CM from shHDAC1-transduced CMCs contained more cytokines involved in cell growth/differentiation and more efficiently promoted endothelial cell proliferation and tube formation compared with CM from shNT. After evaluating key cytokines previously implicated in cell-therapy-mediated cardiac repair, we found that basic fibroblast growth factor was significantly upregulated in shHDAC1-transduced CMCs. Furthermore, shRNA-mediated knockdown of basic fibroblast growth factor in HDAC1-depleted CMCs inhibited the effects of shHDAC1 CM in promoting endothelial proliferation and tube formation-indicating that HDAC1 depletion activates CMC proangiogenic paracrine signaling in a basic fibroblast growth factor-dependent manner. CONCLUSIONS: These results reveal a hitherto unknown role for HDAC1 in the modulation of CMC cytokine secretion and implicate the targeted inhibition of HDAC1 in CMCs as a means to enhance paracrine-mediated neovascularization in cardiac cell therapy applications.

Analysis of transcriptional activities of angiogenic biomarkers during intrauterine complications leading to preterm birth.

OBJECTIVE: Pre-eclampsia, growth retardation and preterm delivery are the most common reasons leading to increased maternal and perinatal mortality. The increased expression of hypoxia induced factors, such as HIF-1, triggers the overexpression of anti-angiogenic genes. The aim of this study was to determine the transcriptional activity of individual pro- and anti-angiogenic markers (VEGF, HIF-1, sEng, Flt-1, PlGF-1) in maternal blood samples from patients with spontaneous preterm labor, preterm labor in combination with pre-eclampsia and fetal growth restriction in comparison with physiologically terminated pregnancies. PATIENTS AND METHODS: The transcriptional activity of specific genes was detected from the blood of patients using the chromatin immunoprecipitation capture method coupled with quantitative real-time PCR. RESULTS: The maximum differences in mRNA levels of PlGF-1 and VEGF-A were detected in two groups: the group of normal-term birth with complications and the group of preterm labor with complications (both significantly lower than the control, p < 0.001). In contrast, a marked increase of mRNA levels was found in the same groups of patients for the HIF-1, endoglin and Flt-1 genes (p < 0.001). CONCLUSIONS: According to our results, we can conclude that increased oxidative stress, increasing the expression levels of anti-angiogenic genes and reduction of the transcriptional activity of pro-angiogenic genes can provide additional information during diagnostics of pathological complications of labor.

High Expression of Angiogenic Factor with G-Patch and FHA Domain1 (AGGF1) Predicts Poor Prognosis in Gastric Cancer.

BACKGROUND Angiogenic factor with G-patch and FHA domain1 (AGGF1 or VG5Q) is a newly identified human angiogenic factor. The aim of this study was to explore AGGF1 expression level in gastric cancer and detect its correlation with the prognosis. MATERIAL AND METHODS Immunohistochemistry was performed to detect AGGF1 level in gastric cancer and its adjacent noncancerous samples of 198 cases, and the relationships among the expression levels of AGGF1, vascular endothelial growth factor (VEGF), and prognosis were analyzed. RESULTS Expression of AGGF1 in gastric cancer samples was significantly higher than that in adjacent noncancerous samples (P<0.001). The overall survival rate (OS) of patients with high AGGF1 expression was significantly lower than that of patients with low AGGF1 expression (P=0.000). The Cox model analysis demonstrated that expression of AGGF1 was an independent biomarker for prediction of patients' survival in gastric cancer. CONCLUSIONS High expression of AGGF1 predicts poor prognosis in gastric cancer patients. AGGF1 can be used as an independent factor to predict postoperative survival of patients with gastric cancer.

Prenatal stress-induced increases in hippocampal von Willebrand factor expression are prevented by concurrent prenatal escitalopram.

Prenatal stress has been linked to deficits in neurological function including deficient social behavior, alterations in learning and memory, impaired stress regulation, and susceptibility to adult disease. In addition, prenatal environment is known to alter cardiovascular health; however, limited information is available regarding the cerebrovascular consequences of prenatal stress exposure. Vascular disturbances late in life may lead to cerebral hypoperfusion which is linked to a variety of neurodegenerative and psychiatric diseases. The known impact of cerebrovascular compromise on neuronal function and behavior highlights the importance of characterizing the impact of stress on not just neurons and glia, but also cerebrovasculature. Von Willebrand factor has previously been shown to be impacted by prenatal stress and is predictive of cerebrovascular health. Here we assess the impact of prenatal stress on von Willebrand factor and related angiogenic factors. Furthermore, we assess the potential protective effects of concurrent anti-depressant treatment during in utero stress exposure on the assessed cerebrovascular endpoints. Prenatal stress augmented expression of von Willebrand factor which was prevented by concurrent in utero escitalopram treatment. The functional implications of this increase in von Willebrand factor remain elusive, but the presented data demonstrate that although prenatal stress did not independently impact total vascularization, exposure to chronic stress in adulthood decreased blood vessel length. In addition, the current study demonstrates that production of reactive oxygen species in the hippocampus is decreased by prenatal exposure to escitalopram. Collectively, these findings demonstrate that the prenatal experience can cause complex changes in adult cerebral vascular structure and function.

Haploinsufficiency of Klippel-Trenaunay syndrome gene Aggf1 inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin.

Aggf1 is the first gene identified for Klippel-Trenaunay syndrome (KTS), and encodes an angiogenic factor. However, the in vivo roles of Aggf1 are incompletely defined. Here we demonstrate that Aggf1 is essential for both physiological angiogenesis and pathological tumour angiogenesis in vivo. Two lines of Aggf1 knockout (KO) mice showed a particularly severe phenotype as no homozygous embryos were observed and heterozygous mice also showed embryonic lethality (haploinsufficient lethality) observed only for Vegfa and Dll4. Aggf1+/- KO caused defective angiogenesis in yolk sacs and embryos. Survived adult heterozygous mice exhibit frequent haemorrhages and increased vascular permeability due to increased phosphorylation and reduced membrane localization of VE-cadherin. AGGF1 inhibits VE-cadherin phosphorylation, increases plasma membrane VE-cadherin in ECs and in mice, blocks vascular permeability induced by ischaemia-reperfusion (IR), restores depressed cardiac function and contraction, reduces infarct sizes, cardiac fibrosis and necrosis, haemorrhages, edema, and macrophage density associated with IR. Mechanistically, AGGF1 promotes angiogenesis by activating catalytic p110α subunit and p85α regulatory subunit of PI3K, leading to activation of AKT, GSK3ß and p70S6K. AKT activation is significantly reduced in heterozygous KO mice and isolated KO ECs, which can be rescued by exogenous AGGF1. ECs from KO mice show reduced capillary angiogenesis, which is rescued by AGGF1 and AKT. Tumour growth/angiogenesis is reduced in heterozygous mice, which was associated with reduced activation of p110α, p85α and AKT. Together with recent identification of somatic mutations in p110α (encoded by PIK3CA), our data establish a potential mechanistic link between AGGF1 and PIK3CA, the two genes identified for KTS.

Phase I study of multi-gene cell therapy in patients with peripheral artery disease.

UNLABELLED: Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 10(7), Cohort 2: 2 × 10(7), Cohort 3: 5 × 10(7), Cohort 4: 7 × 10(7)). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle-brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00390767.

Vascular Endothelial Growth Factor A Regulates the Secretion of Different Angiogenic Factors in Lung Cancer Cells.

Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention.

Nuclear vasohibin-2 promotes cell proliferation by inducing G0/G1 to S phase progression.

As a member of the vasohibin (VASH2) family, VASH2 is localized intracellularly as a nuclear and cytoplasmic type. Cytoplasmic VASH2 is associated with carcinoma angiogenesis and malignant transformation and promotes cancer growth. However, the function of nuclear VASH2 has yet to be investigated. The aim of the present study was to detect the nuclear VASH2 expression profile in human organs and tissues by protein microarray technique. To examine the function of nuclear VASH2, we analyzed the relationship between nuclear VASH2 and Ki-67, and stably constructed VASH2 overexpression and knockdown in LO2 and HepG2 cell lines, based on a previous study in hepatic cells. The study was conducted using bromodeoxyuridine, immunofluorescent staining, western blot analysis and flow cytometry. Nuclear VASH2 was highly expressed in actively dividing cells in normal and cancer tissues. There was a significant positive correlation between nuclear VASH2 and Ki-67, indicating that nuclear VASH2 positively correlated with cell proliferation in normal and cancer tissues. The bromodeoxyuridine (BrdU) proliferation test showed that nuclear VASH2 increased the S-phase population and promoted cell proliferation, while VASH2 knockdown reduced BrdU absorbance. Cell cycle analysis revealed that nuclear VASH2 overexpression increased the S-phase population in LO2 and HepG2 cells, while nuclear VASH2 knockdown reduced the S-phase population and increased the G0/G1 population. The findings of this study challenge the classic view of VASH2, which was previously reported as an angiogenesis factor. Furthermore, to the best of our knowledge, these results are the first clinical data indicating that nuclear VASH2, but not cytoplasmic VASH2, promotes cell proliferation by driving the cell cycle from the G0/G1 to S phase.

Suppressed inflammatory gene expression during human hypertrophic scar compared to normotrophic scar formation.

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar (HTscar) and not to normotrophic scar (NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix (ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52 weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes (TNFα, IL-1α, IL-1RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL-10) and an extended increased expression of ECM-related genes (PLAU, Col3A1, TGFß3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti-inflammatory scar therapies.

Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice.

Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.