Expression of pigment-epithelium-derived factor during kidney development and aging

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development. Ana Luisa Pina and Marion Kubitza contributed equally to this work.     

Expression of soluble, biologically active recombinant human endostatin in Escherichia coli

Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

Chondromodulin-I and tenomodulin: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues

In tissues and/or organs of mesenchymal origin, the vasculature is usually well developed. However, there are certain hypovascular tissues that exhibit powerful anti-angiogenic resistance, implying the presence of tissue-type specific inhibitors of angiogenesis. Hyaline cartilage is one example, and several anti-angiogenic factors have been purified from cartilage. We previously identified chondromodulin-I (ChM-I) as a tissue-specific inhibitor of angiogenesis in fetal bovine cartilage. ChM-I is specifically expressed in the avascular regions of the growth-plate and cartilaginous bone rudiments in embryos. Recently, we cloned a novel type II transmembrane protein, tenomodulin (TeM), having a domain homologous to ChM-I at its C-terminus. TeM turned out to be expressed specifically in other hypovascular structures in the mesenchyme, such as the epimysium, tendon, and ligaments. In this overview, we discuss the structural characteristics of this class of anti-angiogenic molecules and their pathophysiological role in the control of vascularity.     

Synthesis of a 6(V)-sulfated mannopentasaccharide analogue related to PI-88

An efficient and convergent synthesis of a regioselectively 6(V)-sulfated mannopentasaccharide derivative 1c, octyl 6-O-sulfo-alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranosyl-(1-->3)-alpha-d-mannopyranosyl-(1-->3)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranoside, was achieved by a '3 + 2' strategy. The target was designed to mimic the promising anticancer agent PI-88 and was obtained from the building blocks, octyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, allyl 2,4,6-tri-O-benzoyl-3-O-(4-methoxybenzyl)-alpha-D-mannopyranoside, and 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (11), under TMSOTf-catalyzed glycosylation conditions. Compound 1c displays a mild anti-angiogenic activity based on a chorioallantoic membrane (CAM) model study.     

Metabolic effects of anti-angiogenic therapy in tumors

Anti-angiogenic therapy has recently been added to the panel of cancer therapeutics, but predictive biomarkers of response are still not available. In animal models, anti-angiogenic therapy causes tumor starvation by increasing hypoxia and impairing nutrients supply. It is thus conceivable that angiogenesis inhibition causes remarkable metabolic perturbations in tumors, although they remain largely uncharted. We review here recent acquisitions about metabolic effects of angiogenesis blockade in tumors and discuss the possibility that some metabolic features of tumor cells - i.e. their dependency from glucose as primary energy substrate - might affect tumor responses to anti-VEGF treatment.     

Differential actions of VEGF-A isoforms on perichondrial angiogenesis during endochondral bone formation

During endochondral bone formation, vascular invasion initiates the replacement of avascular cartilage by bone. We demonstrate herein that the cartilage-specific overexpression of VEGF-A₁₆₄ in mice results in the hypervascularization of soft connective tissues away from cartilage. Unexpectedly, perichondrial tissue remained avascular in addition to cartilage. Hypervascularization of tissues similarly occurred when various VEGF-A isoforms were overexpressed in the chick forelimb, but also in this case perichondrial tissue and cartilage were completely devoid of vasculature. However, following bony collar formation, anti-angiogenic properties in perichondrial tissue were lost and perichondrial angiogenesis was accelerated by VEGF-A₁₄₆, VEGF-A₁₆₆, or VEGF-A₁₉₀. Once the perichondrium was vascularized, osteoclast precursors were recruited from the circulation and the induction of MMP9 and MMP13 can be observed in parallel with the activation of TGF-β signaling. Neither perichondrial angiogenesis nor the subsequent cartilage vascularization was found to be accelerated by the non-heparin-binding VEGF-A₁₂₂ or by the VEGF-A₁₆₆ΔE₁₆₂-R₁₆₆ mutant lacking a neuropilin-binding motif. Hence, perichondrial angiogenesis is a prerequisite for subsequent cartilage vascularization and is differentially regulated by VEGF-A isoforms.     

氢氧化钠瘤内注射通过抗血管生成作用抑制肝癌生长的机制研究

目的:研究氢氧化钠溶液瘤内注射对肝癌的生长抑制作用并探索其机制。方法:对SMMU-LTNM肝癌裸鼠皮下模型进行2%浓度的氢氧化钠溶液瘤内注射,检测肿瘤组织微血管密度、HIF-1α和VEGF的表达情况。结果:与生理盐水瘤内注射组相比,氢氧化钠显著抑制肿瘤生长(P<0.01)、降低肿瘤微血管密度(P=0.01)、抑制肿瘤组织HIF-1α和VEGF的表达(P=0.02和P=0.01)。结论:氢氧化钠瘤内注射可有效抑制肝癌生长,主要机制可能是抗血管生成作用。     

Surgery and angiogenesis

Surgery may be regarded as an angiogenesis-inducing condition since it evokes the release of many angiogenic factors. Regarding the mechanistic overlap between tumor-associated neovascularisation and (physiological) angiogenesis in response to injury and hypoxia, surgery may promote the uncontrolled growth of residual dormant tumor cells. With the advent of anti-angiogenic agents, surgeons will be faced with more patients undergoing surgery for primary and secondary tumors under anti-angiogenic treatment. This could present problems with regard to angiogenesis-dependent phenomena such as wound repair, healing of intestinal anastomoses and liver regeneration. In this review we will discuss these matters from a biomedical and clinical point of view.     

Protein expression of pigment-epithelium-derived factor in rat cochlea

Pigment-epithelium-derived factor (PEDF) is a 50-kDa glycoprotein with well-recognised expression in various mammalian organs showing diverse (e.g. anti-angiogenic and neuroprotective) activities. However, at present, no information is available regarding the potential function of this cytokine in the inner ear. As a first approach to investigating whether PEDF is involved in cochlear function, we have explored its protein expression in the rat cochlea by immunocytochemistry. Our results show that PEDF expression in the cochlea is most prominent in the basilar membrane below the organ of Corti, in the lateral wall (especially in the stria vascularis), in ganglion neurons, and in the endothelia of blood vessels. Our findings on its distribution in the cochlea suggest that PEDF in the basilar membrane prevents blood vessel formation that would disturb cochlear micromechanics and would interfere with the mechano-electrical transduction in the organ of Corti. In cochlear ganglion neurons, PEDF might serve a neuroprotective function possibly protecting these neurons from excessive glutamate released by the inner hair cells. Our data constitute the first report on the morphological protein distribution of this multifunctional molecule in the rat cochlea and suggest its role in important functions of the internal ear. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Modeling the VEGF–Bcl-2–CXCL8 Pathway in Intratumoral Agiogenesis

Recent experiments show that vascular endothelial growth factor (VEGF) is the crucial mediator of downstream events that ultimately lead to enhanced endothelial cell survival and increased vascular density within many tumors. The newly discovered pathway involves up-regulation of the anti-apoptotic protein Bcl-2, which in turn leads to increased production of interleukin-8 (CXCL8). The VEGF–Bcl-2–CXCL8 pathway suggests new targets for the development of anti-angiogenic strategies including short interfering RNA (siRNA) that silence the CXCL8 gene and small molecule inhibitors of Bcl-2. In this paper, we present and validate a mathematical model designed to predict the effect of the therapeutic blockage of VEGF, CXCL8, and Bcl-2 at different stages of tumor progression. In agreement with experimental observations, the model predicts that curtailing the production of CXCL8 early in development can result in a delay in tumor growth and vascular development; however, it has little effect when applied at late stages of tumor progression. Numerical simulations also show that blocking Bcl-2 up-regulation, either at early stages or after the tumor has fully developed, ensures that both microvascular and tumor cell density stabilize at low values representing growth control. These results provide insight into those aspects of the VEGF–Bcl-2–CXCL8 pathway, which independently and in combination, are crucial mediators of tumor growth and vascular development. Continued quantitative modeling in this direction may have profound implications for the development of novel therapies directed against specific proteins and chemokines to alter tumor progression.