ApoB-containing lipoproteins in apoE-deficient mice are not metabolized by the class B scavenger receptor BI

The scavenger receptor class B type I (SR-BI) recognizes a broad variety of lipoprotein ligands, including HDL, LDL, and oxidized LDL. In this study, we investigated whether SR-BI plays a role in the metabolism of cholesterol-rich lipoprotein remnants that accumulate in apolipoprotein E (apoE)(-/-) mice. These particles have an unusual apolipoprotein composition compared with conventional VLDL and LDL, containing mostly apoB-48 as well as substantial amounts of apoA-I and apoA-IV. To study SR-BI activity in vivo, the receptor was overexpressed in apoE(-/-) mice by adenoviral vector-mediated gene transfer. An approximately 10-fold increase in liver SR-BI expression resulted in no detectable alterations in VLDL-sized particles and a modest depletion of cholesterol in intermediate density lipoprotein/LDL-sized lipoprotein particles. This decrease was not attributable to altered secretion of apoB-containing lipoproteins in SR-BI-overexpressing mice. To directly assess whether SR-BI metabolizes apoE(-/-) mouse lipoprotein remnants, in vitro assays were performed in both CHO cells and primary hepatocytes expressing high levels of SR-BI. This analysis showed a remarkable deficiency of these particles to serve as substrates for selective lipid uptake, despite high-affinity, high-capacity binding to SR-BI. Taken together, these data establish that SR-BI does not play a direct role in the metabolism of apoE(-/-) mouse lipoprotein remnants.     

Linc-ROR facilitates progression and angiogenesis of hepatocellular carcinoma by modulating DEPDC1 expression

Linc-ROR have been well-demonstrated to play important roles in cancer progression and angiogenesis. However, the underlying oncogenic mechanism of Linc-ROR in hepatocellular carcinoma is poorly understood. In this study, we demonstrate that Linc-ROR plays an oncogenic role in part through its positive regulation of DEPDC1 expression. Mechanistically, Linc-ROR acts as competing endogenous RNA to stabilize DEPDC1 mRNA and regulates DEPDC1 mRNA stability by binding HNRNPK. Thus, these findings suggest that function of Linc-ROR-mediated DEPDC1 could predispose hepatocellular carcinoma patients to progression and angiogenesis, and may serve as a potential target for anticancer therapies.Subject terms: Gastrointestinal cancer, Cancer     

Histamine synergistically promotes bFGF‐induced angiogenesis by enhancing VEGF production via H1 receptor

Histamine, a major mediator present in mast cells that is released into the extracellular milieu upon degranulation, is well known to possess a wide range of biological activities in several classic physiological and pathological processes. However, whether and how it participates in angiogenesis remains obscure. In the present study, we observed its direct and synergistic action with basic fibroblast growth factor (bFGF), an important inducer of angiogenesis, on in vitro angiogenesis models of endothelial cells. Data showed that histamine (0.1, 1, 10 µM) itself was absent of direct effects on the processes of angiogenesis, including the proliferation, migration, and tube formation of endothelial cells. Nevertheless, it could concentration‐dependently enhance bFGF‐induced angiogenesis as well as production of vascular endothelial growth factor (VEGF) from endothelial cells. The synergistic effect of histamine on VEGF production could be reversed by pretreatments with diphenhydramine (H1‐receptor antagonist), SB203580 (selective p38 mitogen‐activated protein kinase (MAPK) inhibitor) and L ‐NAME (nitric oxide synthase (NOS) inhibitor), but not with cimetidine (H2‐receptor antagonist) and indomethacin (cyclooxygenase (COX) inhibitor). Moreover, histamine could augment bFGF‐incuced phosphorylation and degradation of IκBα, a key factor accounting for the activation and translocation of nuclear factor κB (NF‐κB) in endothelial cells. These findings indicated that histamine was able to synergistically augment bFGF‐induced angiogenesis, and this action was linked to VEGF production through H1‐receptor and the activation of endothelial nitric oxide synthase (eNOS), p38 MAPK, and IκBα in endothelial cells. J. Cell. Biochem. 114: 1009–1019, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of angiogenesis and tumor growth by p110 alpha and AKT1 via VEGF expression

Recent studies demonstrate that PI3K activation and PTEN mutation are frequently found in many human cancer cells and tissues. However, the mechanism of PI3K signaling in human cancer tumorigenesis remains to be elucidated. In this study we specifically downregulated p110alpha expression in ovarian cancer cells using siRNA interference. We found that p110alpha downregulation greatly decreased ovarian tumor growth and angiogenesis, and that p110alpha siRNA inhibited VEGF expression through decreasing hypoxia-inducible factor 1alpha expression in both ovarian cancer cells and tumor tissues. To determine the downstream targets of PI3K in regulating tumor growth and angiogenesis, we find that AKT1 is a major downstream mediator for regulating tumor growth, angiogenesis, and VEGF expression. These data show that p110alpha and AKT1 play an important role in tumor growth by inducing angiogenesis and by increasing HIF-1alpha and VEGF expression. This work provides a better understanding of the molecular mechanism of human cancer induced by the activation of PI3K signaling.     

VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

Correlation between VEGF expression and angiogenesis in breast carcinoma

OBJECTIVE: To analyze the role of vascular endothelial growth factor (VEGF) secreted by tumor cells in angiogenesis of breast carcinoma using image morphometry. STUDY DESIGN: Thirty-four cases of node-negative breast carcinoma were used in the study. There were 6 grade 1, 20 grade 2 and 8 grade 3 tumors. For each case, 2 consecutive sections from the same block were cut. Immunostaining for VEGF and CD31 was carried out, and areas of highest staining density were marked. Those marked "hot spots" for CD31 and VEGF for each case were subsequently compared morphometrically. The area and intensity of immunostaining on each slide were also scored. RESULTS: The total scores for VEGF and CD31 were 5.15 and 3.79, respectively. All 34 cases showed cytoplasmic positivity for VEGF within the tumor cells. The average number of hot spots for VEGF and CD31 were 2.41 and 2.47, respectively, and the average number of hot spots that matched between these 2 groups were 0.79. Statistical analysis using Pearson's coefficient of correlation showed no significant match between the hot spots for CD31 and VEGF. Also, there was no significant difference between the total scores of CD31 and VEGF. CONCLUSION: VEGF is expressed in most breast carcinomas. However, the lack of topographic correlation between microvessel density and VEGF expression supports the notion that multiple angiogenic factors may play a role along with VEGF in the angiogenic process.     

Soy protein,casein, and zein regulate histidase gene expression by modulating serum glucagon

Glucagon has been postulated as an important physiological regulator of histidase (Hal) gene expression; however, it has not been demonstrated whether serum glucagon concentration is associated with the type and amount of protein ingested. The purpose of the present work was to study the association between hepatic Hal activity and mRNA concentration in rats fed 18 or 50% casein, isolated soy protein, or zein diets in a restricted schedule of 6 h for 10 days, and plasma glucagon and insulin concentrations. On day 10, five rats of each group were killed at 0900 (fasting), and then five rats were killed after being given the experimental diet for 1 h (1000). Rats fed 50% casein or soy diets showed higher Hal activity than the other groups studied. Rats fed 50% zein diets had higher Hal activity than rats fed 18% casein, soy, or zein diets, but lower activity than rats fed 50% casein or soy diets. Hal mRNA concentration followed a similar pattern. Hal activity showed a significant association with serum concentrations of glucagon. Serum glucagon concentration was significantly correlated with protein intake. Thus the type and amount of protein consumed affect Hal activity and expression through changes in serum glucagon concentrations.     

Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.     

Human EP3(I) prostanoid receptor induces VEGF and VEGF receptor-1 mRNA expression

A critical event in tumor development is the formation of new blood vessels to provide oxygen, nutrients and growth factors to the rapidly growing cancer cells. This process of angiogenesis is complex, however, it is well established that vascular endothelial growth factor (VEGF)-mediated signaling is an important early event. Knockout mice studies have implicated the EP3 receptor in tumor development and angiogenesis; however, the signaling mechanism involved with this effect is unclear. We now show that stimulation of the EP3_I isoform of the human EP3 receptor with prostaglandin E₂ increases the mRNA expression of both VEGF and its cognate receptor VEGF receptor-1 (VEGFR-1). These inductions by the EP3_I receptor involve the sequential activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Up-regulation of VEGF and VEGFR-1 mRNA by the human EP3_I receptor has not been previously reported and further strengthen the role of this receptor in tumor-associated angiogenesis.     

Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.     

Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and pRSV-Raf-BXB, also inhibited pRSV-Raf-BXB-induced sIL-1Ra promoter activity, suggesting that inductions of sIL-1Ra promoter activity by LPS and Raf-1 actually occur by mutually antagonistic mechanisms. In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-1 responses localized to different regions of the sIL-1Ra promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL-1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway.     

Effect of modulating cardiac A1 adenosine receptor expression on protection with ischemic preconditioning

Activation of A(1) adenosine receptors (A(1)ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological A(1)AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified A(1)AR expression to define the role of A(1)AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted (A(1)KO(-/-)) or single-copy (A(1)KO(+/-)) A(1)AR, and transgenic mice (A(1)TG) with increased cardiac A(1)AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, A(1)KO(+/-), and A(1)KO(-/-) mice was (in % of risk region) 58 +/- 3, 60 +/- 4, and 61 +/- 2, respectively, and was less in A(1)TG mice (39 +/- 4, P < 0.05). A strong correlation was observed between A(1)AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 +/- 3, P < 0.05 vs. WT), A(1)KO(+/-) + IPC (48 +/- 4, P < 0.05 vs. A(1)KO(+/-)), and A(1)TG + IPC mice (24 +/- 2, P < 0.05 vs. A(1)TG). However, IPC did not decrease IF in A(1)KO(-/-) + IPC mice (63 +/- 2). In addition, A(1)KO(-/-) hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that A(1)ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of A(1)AR gene expression.     

IL-17A differentially regulates corneal vascular endothelial growth factor (VEGF)-A and soluble VEGF receptor 1 expression and promotes corneal angiogenesis after herpes simplex virus infection

Ocular infection with HSV causes corneal neovascularization (CV), an essential step in the pathogenesis of the blinding immunoinflammatory lesion stromal keratitis. The infection results in IL-17A production, which contributes to CV in ways that together serve to shift the balance between corneal concentrations of vascular endothelial growth factor A (VEGF-A) and the soluble vascular endothelial growth factor receptor 1 molecule, which binds to VEGF-A and blocks its function (a so-called VEGF trap). Accordingly, animals lacking responses to IL-17A signaling, either because of IL-17 receptor A knockout or wild-type animals that received neutralizing mAb to IL-17A, had diminished CV, compared with controls. The procedures reduced VEGF-A protein levels but had no effect on the levels of soluble vascular endothelial growth factor receptor 1. Hence the VEGF trap was strengthened. IL-17A also caused increased CXCL1/KC synthesis, which attracts neutrophils to the inflammatory site. Neutrophils further influenced the extent of CV by acting as an additional source of VEGF-A, as did metalloproteinase enzymes that degrade the soluble receptor, inhibiting its VEGF-blocking activity. Our results indicate that suppressing the expression of IL-17A, or increasing the activity of the VEGF trap, represents a useful approach to inhibiting CV and the control of an ocular lesion that is an important cause of human blindness.