CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis.     

Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells

Peptidoglycan (PG) is the major component of the cell wall of gram-positive bacteria. In vitro, PG isolated from conventional bacterial cultures can induce secretion of proinflammatory cytokines by human monocytes, indicating that PG may be involved in immune responses against infections by gram-positive bacteria. To investigate the biologic activity of PG in human tissues, an improved method was developed to isolate significant amounts of PG from sterile human spleen tissue. Biochemical analysis demonstrated that PG isolated from human spleen is largely intact. Human whole blood cell cultures were able to produce the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 and -6 after stimulation with PG isolated from human spleen. Cytokine induction was not sensitive to inhibition by polymyxin B, in contrast to lipopolysaccharide. Collectively, the data show that intact PG in sterile human tissue is biologically active and may induce local proinflammatory cytokine production.     

Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

AIMS: Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined. METHODS AND RESULTS: mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha, delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation. CONCLUSIONS: PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC alpha-dependent positive feedback mechanism.     

Transforming growth factor-beta stimulates vascular endothelial growth factor production by folliculostellate pituitary cells

TGF-beta isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-beta has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-beta. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-beta1, -beta2, and -beta3. They also express TGF-beta receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-betabinding and signaling. Stimulation of TtT/GF cells with either TGF-beta1 or TGF-beta3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-beta isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-beta-induced VEGF production by cycloheximide suggest that TGF-beta induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-beta1 and -beta3 stimulated VEGF production. TGF-beta stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.     

Improved fat transplantation survival by using the conditioned medium of vascular endothelial growth factor transfected human adipose-derived stem cells

Autologous fat transplantation has been applied widely in clinic. However, the low survival rate is still a problem to be solved. Studies shows that the human adipose-derived stem cells (ADSCs) transfected by vascular endothelial growth factor (VEGF) can improve the survival rate of autologous fat transplantation. Our study is to evaluate the effects of the conditioned medium of VEGF-transfected human adipose-derived stem cells (VEGF-ADSCs-CM) on fat transplantation. ADSCs were isolated and transfected with MOI = 40. The study was divided into three groups, VEGF-ADSCs-CM group, normal-ADSCs-CM group and control group. The conditioned media for VEGF-ADSCs-CM group and normal-ADSCs-CM group were collected, and then mixed with fat, with the mixtures being injected into the back of nude mice. On 4, 7, 15, 30, 60 days after transplantation, the grafts were evaluated on the wet weight, histology, ELISA and western blot. As the results revealed, the survival rate of VEGF-ADSCs-CM group was highest with the best fat cell morphology, and the VEGF secretion of VEGF-ADSCs-CM group was also highest. Therefore, our study demonstrates that VEGF-ADSCs-CM can improve the survival rate of fat transplantation effectively, and VEGF-ADSCs-CM can be regarded as an effective assisted method for fat transplantation.     

Hormonal influences on the secretion of steroid-binding proteins by a human hepatoma-derived cell line

We examined the influence of numerous substances on the secretion of corticosteroid-binding globulin (CBG) and testosterone-estradiol binding globulin (TeBG) by a human hepatoma-derived cell line. Thyroxine, at physiologic concentrations, resulted in an increased secretion of TeBG but not CBG. Estrogens, antiestrogens, and androgens were without effect on either of these binding proteins.     

Development of angiogenic cell and gene therapy by transplantation of umbilical cord blood with vascular endothelial growth factor gene.

Endothelial progenitor cells (EPCs) are present in the mononuclear cells (MNCs) of umbilical cord blood and peripheral blood. To establish the efficiency of angiogenic cell and gene therapies, we transfected the human vascular endothelial growth factor (hVEGF) gene into cord blood MNCs to enhance endothelialization. MNCs from cord blood and peripheral blood were isolated and transfected with pCR3 expressing hVEGF165 or GFP by the Hemagglutinating Virus of Japan (HVJ)-envelope and the cells were cultured in endothelium basal medium-2. The number of attached cells from cord blood was higher than that from peripheral blood. Attached cells expressed Flk-1, VE-cadherin, PECAM-1, CD34, and Tie-2. The increase in the number of attached cells was transient with the transfection of vascular endothelial growth factor (VEGF) gene early in the experimental period. Flt-1 mRNA was not expressed early in the culture period, but was expressed at 2 weeks after separation. VEGF gene transfer into MNCs at 12 days after separation, i.e., when Flt-1 mRNA was expressed continuously, increased the number of attached cells. We evaluated the effects of the transplantation of cord blood MNCs expressing the hVEGF gene on regional blood flow in an ischemic area in a rat model of chronic hindlimb ischemia. Blood flow was significantly improved in nude rats that received transplanted control MNCs. Transplantation of cord blood MNCs transfected with the hVEGF gene yielded greater improvements in blood flow. These results indicate that the hVEGF gene enhances endothelialization of EPCs, and that the transplantation of cord blood MNCs transfected with the VEGF gene may be feasible for the treatment of ischemic diseases as a type of angiogenic cell and gene therapy.     

Roxithromycin specifically inhibits development of collagen induced arthritis and production of proinflammatory cytokines by human T cells and macrophages

OBJECTIVE: Roxithromycin (RXM) is a macrolide antibiotic that is effective in treatment of chronic lower respiratory tract diseases including diffuse panbronchiolitis and bronchial asthma. Its mechanism of action apart from its antibacterial action remains unclear. To determine the mechanism of action of RXM, we evaluated the effect of RXM on T cell functions and the inflammatory responses in mice with collagen induced arthritis (CIA). METHODS: T cell proliferation, cytokine production by T cells stimulated through CD28, CD26, or PMA with or without anti-CD3 Mab, cytokine production by macrophages stimulated with lipopolysaccharide, and transendothelial migration of T cells were analyzed in the presence or absence of various concentrations of RXM. We evaluated the effect of RXM treatment in collagen induced arthritis in mice. RESULTS: RXM did not affect the production of Th1-type and Th2-type cytokines, whereas it specifically inhibited production of proinflammatory cytokines such as tumor necrosis factor-a and interleukin 6 (IL-6) by T cells and macrophages. RXM inhibited T cell migration. We found that RXM treatment of mice with CIA reduced the severity of arthritis and serum level of IL-6, as well as leukocyte migration into the affected joints and destruction of bones and cartilage. CONCLUSION: Our findings strongly suggest that RXM may be useful for the therapy of rheumatoid arthritis as well as other inflammatory diseases such as Crohn's disease.     

The embryonic angiogenic factor Del1 accelerates tumor growth by enhancing vascular formation

Del1 is a unique alpha v beta 3 integrin ligand that is produced by endothelial cells, and thus provides an autocrine signaling pathway in this cell type. It is expressed transiently in the embryo and mediates cell attachment, migration, and activation of cytoplasmic signaling molecules in focal contacts. Del1 also activates angiogenesis in the chick chorioallantoic membrane assay. Reexpression of this embryonic signaling molecule has now been documented in naturally occurring human tumors, where it is expressed by both tumor cells and angiogenic endothelial cells, suggesting that Del1 is important in mediating angiogenesis under pathophysiological conditions in the adult. To investigate the role of Del1 in tumor growth and angiogenesis, human 143B osteosarcoma cells and murine Lewis lung carcinoma cells were engineered to express Del1 and compared to control transfectants for their ability to produce tumors in nude or syngeneic mice, respectively. Del1 expressing tumors showed a two- to fourfold increase in capillary density and an accelerated rate of growth. Expression of Del1 also correlated with a decrease in apoptosis in tumor cells in vivo. Taken together, these data suggest that Del1 acts as an angiogenic factor in the context of solid tumor formation and that this increase in vascularization accelerates tumor growth through decreased apoptosis.     

The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin

Nucleolin, originally described as a nuclear protein, was recently found to be expressed on the surface of endothelial cells during angiogenic. However, the functions of cell-surface nucleolin in angiogenic remain mysterious. Here we report that upon endothelial cells adhering to extracellular matrix components, vascular endothelial growth factor (VEGF) mobilizes nucleolin from nucleus to cell surface. Functional blockage or down-regulation of the expression of cell-surface nucleolin in endothelial cells significantly inhibits the migration of endothelial cells and prevents capillary-tubule formation. Moreover, nonmuscle myosin heavy chain 9 (MyH9), an actin-based motor protein, is identified as a nucleolin-binding protein. Subsequent studies reveal that MyH9 serves as a physical linker between nucleolin and cytoskeleton, thus modulating the translocation of nucleolin. Knocking down endogenous MyH9, specifically inhibiting myosin activity, or overexpressing functional deficient MyH9 disrupts the organization of cell-surface nucleolin and inhibits its angiogenic function. These studies indicate that VEGF, extracellular matrix, and intracellular motor protein MyH9 are all essential for the novel function of nucleolin in angiogenic.     

Production of insulin-like growth factor binding proteins by human ovarian carcinoma cells

Cells of the human ovarian carcinoma lines EFO-21, EFO-27, MFO-35 and MFO-36 secrete binding proteins for insulin-like growth factors (IGFBPs) into their culture media. By sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting, seven groups of IGFBPs with molecular masses of 25, 30 (doublet), 34, 37, 40, 45, and 50 kDa were observed, depending on the cell line under investigation. By Northern blot analyses using cDNAs or oligonucleotides specific for the six types of IGFBP (IGFBP-1 to IGFBP-6), mRNA for all IGFBPs tested except for IGFBP-1 could be detected in the ovarian carcinoma cell extracts. In detail, analysis of EFO-21 protein products by SDS-PAGE yielded IGFBPs of 25, 34, and 50 kDa; extracts of EFO-21 cells contained mRNAs for IGFBP-2, -3, -4, and -6. EFO-27 cells produced IGFBPs of 40 kDa and 45 kDa as determined by SDS-PAGE, and mRNAs for IGFBP-3, -4, and -6 were detected. In the conditioned medium of MFO-35 cells, IGFBPs of 25, 30 (doublet), 34, 37, 40, and 45 kDa were observed by SDS-PAGE, while mRNAs for the five proteins IGFBP-2 to IGFBP-6 were found. MFO-36 cells produced IGFBPs of 34 kDa and 50 kDa as determined by SDS-PAGE, and the cells expressed mRNAs for IGFBP-2, -3, -4, and -6. In relation to published molecular mass data of the known IGFBPs, the size of the secreted proteins could be correlated to the mRNA patterns expressed by the ovarian carcinoma cells. It is concluded that ovarian carcinoma cells frequently express IGFBP-3, -4, and -6 and, to a lesser extent, IGFBP-2; the expression of IGFBP-5 appears as a rather rare event, while IGFBP-1 was not found to be expressed in ovarian carcinoma cells.Abbreviations BSA bovine serum albumin - FCS fetal bovine serum - PBS phosphate-buffered saline - SCC citrate-buffered saline - SFM serum-free medium - TRIS/NaCl TRIS-buffered saline Supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB 215, project Al), and by the Bundesministerium für Forschung und Technologie, Bonn, Germany (BMFT grant 01GA8715/9)     

Polymorphism in the Calpain 10 gene influences glucose metabolism in human fat cells

Aims/hypothesis: A common G to A polymorphism (UCSNP-43) in the Calpain 10 gene was recently found to be associated with Type II (non-insulin-dependent) diabetes mellitus and variations in post-absorptive and insulin stimulated glucose metabolism in vivo. We aimed to study the influence of Calpain 10 polymorphism on insulin action in fat cells. Methods: Calpain 10 polymorphism (UCSNP-19, -43 or -63) were set in relation to lipolysis and lipogenesis in isolated subcutaneous adipocytes of 46 apparently healthy non-obese subjects. Results: For UCSNP-43 the G/G genotype had twofold higher basal and insulin stimulated rates as compared with AA/AG genotypes. However, there was no genotype effect on basal or insulin inhibited lipolysis rates in fat cells. The protein amount of GLUT 4 in adipocytes was not influenced by the polymorphism. Fat cells expressed mRNA for the Calpain 10 gene at a relatively high concentration, about 4 amol/μg RNA, which is similar to that of uncoupling protein-2. Neither a UCSNP-19 nor a UCSNP-63 polymorphism in the Calpain 10 gene was found to be associated with basal or insulin-induced adipocyte lipolysis and lipogenesis. None of the polymorphisms influenced body mass index or fasting plasma concentrations of insulin and glucose in 693 non-obese healthy subjects. Conclusions/interpretation: The Calpain 10 gene could be involved in the regulation of glucose metabolism but not lipolysis in human fat cells, although it does not involve adipocyte GLUT-4 protein content. It is possible that the Calpain 10 gene predisposes to diabetes by influencing the glucose metabolism. [Diabetologia (2002) 45: 276–282] Received: 2 July 2001 and in revised form: 28 September 2001