Microvessel formation from mouse aorta is stimulated in vitro by secreted VEGF and extracts from metanephroi

2003 ◽  
Vol 284 (6) ◽  
pp. C1625-C1632 ◽  
Author(s):  
Tetsu Akimoto ◽  
Marc R. Hammerman
Keyword(s):  
Growth Factors ◽  
Organ Culture ◽  
Fusion Protein ◽  
Thoracic Aorta ◽  
Serum Free ◽  
Hypoxic Conditions ◽  
Thoracic Level ◽  
Anti Vegf ◽  
Mouse Aorta

We have demonstrated that during culture under 5% O2, the addition of recombinant human VEGF or FGF2 to mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) stimulates microvessel formation. Here we show that microvessel formation is also stimulated by addition to explants of supernatants obtained from metanephroi grown in serum-free organ culture or of metanephroi extracts. Supernatants and extracts from metanephroi grown under hypoxic conditions are more stimulatory than supernatants/extracts from metanephroi grown in room air. VEGF and FGF2 can be detected by using immunohistochemistry in developing nephrons in the cultured renal anlagen. Metanephroi supernatants contain more VEGF if renal anlagen are grown under hypoxic conditions than if they are grown in room air. Metanephros supernatant-stimulated microvessel formation is completely inhibited by soluble sFlt-1 fusion protein or anti-VEGF antibodies (αVEGF). Extract-stimulated microvessel formation is inhibited by αVEGF or anti-FGF2 antibodies, or both. We conclude that metanephroi produce growth factors including VEGF and FGF that enhance microvessel formation from embryonic thoracic aorta in vitro.

scholarly journals Novel cytokine–antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma

2020 ◽  
Vol 8 (2) ◽  
pp. e001238
Author(s):  
Yaya Chu ◽  
Gaurav Nayyar ◽  
Nang Kham Su ◽  
Jeremy M Rosenblum ◽  
Patrick Soon-Shiong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Fusion Protein ◽  
Natural Killer ◽  
Burkitt Lymphoma ◽  
Ex Vivo ◽  
Specific Binding ◽  
Expression Profiles ◽  
Nsg Mice

BackgroundThe prognosis of patients with relapsed or progressive B cell (CD20+) non-Hodgkin’s lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemoradiotherapy resistance. Novel therapeutic strategies are urgently needed. N-820 is a fusion protein of N-803 (formerly known as ALT-803) to four single-chains of rituximab. This agent has tri-specific binding activity to CD20 and enhanced antibody-dependent cell-mediated cytotoxicity.MethodsWe investigated the anti-tumor combinatorial effects of N-820 with ex vivo expanded peripheral blood natural killer (exPBNK) cells against rituximab-sensitive and rituximab-resistant CD20+ BL in vitro using cytoxicity assays and in vivo using human BL xenografted NOD/SCID/IL2rγnull (NSG) mice. We also investigated the cytokines/chemokines/growth factors released using ELISA and multiplex assay. Gene expression changes were examined using real-time PCR arrays.ResultsN-820 significantly enhanced the expression of NK activating receptors (p<0.001) and the proliferation of exPBNK cells with enhanced Ki67 expression and Stat5 phosphorylation (p<0.001). N-820 significantly enhanced the secretion of cytokines, chemokines, and growth factors including GM-CSF, RANTES, MIP-1B (p<0.001) from exPBNK cells as compared with the combination of rituximab+N-803. Importantly, N-820 significantly enhanced in vitro cytotoxicity (p<0.001) of exPBNK with enhanced granzyme B and IFN-γ release (p<0.001) against BL. Gene expression profiles in exPBNK stimulated by N-820+Raji-2R showed enhanced transcription of CXCL9, CXCL1, CSF2, CSF3, GZMB, and IFNG. Moreover, N-820 combined with exPBNK significantly inhibited rituximab-resistant BL growth (p<0.05) and extended the survival (p<0.05) of BL xenografted NSG mice.ConclusionsOur results provide the rationale for the development of a clinical trial of N-820 alone or in combination with endogenous or ex vivo expanded NK cells in patients with CD20+ B-NHL failing prior rituximab containing chemoimmunotherapy regimens.

scholarly journals Colony growth of human hematopoietic progenitor cells in the absence of serum is supported by a proteinase inhibitor identified as antileukoproteinase.

1996 ◽  
Vol 184 (4) ◽  
pp. 1305-1312 ◽  
Author(s):  
H M Goselink ◽  
J van Damme ◽  
P S Hiemstra ◽  
A Wuyts ◽  
J Stolk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Progenitor Cells ◽  
Conditioned Medium ◽  
Proteinase Inhibitor ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Serum Free ◽  
Accessory Cells

Serum contains many growth factors and nutrients that stimulate colony formation of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of serum, no proliferation of HPCs could be obtained in semisolid medium cultures of partially purified bone marrow cells in the presence of multiple hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade human albumin. This appeared to be due to a suppressive activity induced by monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs. Serum-free conditioned medium from the bladder carcinoma cellline 5637 could replace serum to support the growth of HPCs in these cultures. After gel filtration and reverse-phase high-performance liquid chromatography of 5637 supernatants, this activity could be attributed to a 15-kD protein that was further identified by NH2-terminal sequence analysis as the serine proteinase inhibitor antileukoproteinase (ALP). The growth-supportive activity from the 5637 conditioned medium and the (partially) purified fractions could be completely neutralized by a polyclonal rabbit IgG antibody against human ALP (huALP). Similar supportive effects on the growth of HPC could be obtained in the presence of recombinant huALP. We demonstrated that the COOH-terminal domain of ALP containing the proteinase inhibitory activity was responsible for this effect. alpha-1 proteinase inhibitor was capable of similar support of in vitro HPC growth. These results illustrate that proteinase inhibitors play an important role in the in vitro growth of hematopoietic cells by the neutralization of proteinases produced by bone marrow accessory cells. This may be of particular relevance for in vitro expansion of human hematopoietic stem cells in serum-free media.

Prostatic growth and development are regulated by FGF10

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3693-3701
Author(s):  
A.A. Thomson ◽  
G.R. Cunha
Keyword(s):  
Organ Culture ◽  
Seminal Vesicle ◽  
Growth And Development ◽  
Reproductive Tract ◽  
Mesenchymal Cells ◽  
Female Reproductive Tract ◽  
Ventral Prostate ◽  
Serum Free

We have examined the role of Fibroblast Growth Factor 10 (FGF10) during the growth and development of the rat ventral prostate (VP) and seminal vesicle (SV). FGF10 transcripts were abundant at the earliest stages of organ formation and during neonatal organ growth, but were low or absent in growth-quiescent adult organs. In both the VP and SV, FGF10 transcripts were expressed only in a subset of mesenchymal cells and in a pattern consistent with a role as a paracrine epithelial regulator. In the neonatal VP, FGF10 mRNA was expressed initially in mesenchymal cells peripheral to the peri-urethral mesenchyme and distal to the elongating prostatic epithelial buds. At later stages, mesenchymal cells surrounding the epithelial buds also expressed FGF10 transcripts. During induction of the SV, FGF10 mRNA was present in mesenchyme surrounding the lower Wolffian ducts and, at later stages, FGF10 transcripts became restricted to mesenchymal cells subadjacent to the serosa. We investigated whether the FGF10 gene might be regulated by androgens by analysing the levels of FGF10 transcripts in SV and VP organs grown in serum-free organ culture. While FGF10 transcript levels increased after treatment with testosterone in the SV (but not VP), these changes were not sensitive to anti-androgen treatment, and thus it is likely that FGF10 mRNA was not directly regulated by testosterone. Also, FGF10 mRNA was observed in the embryonic female reproductive tract in a position analogous to that of the ventral prostate in males suggesting that FGF10 is not regulated by androgens in vivo. Recombinant FGF10 protein specifically stimulated growth of Dunning epithelial and BPH1 prostatic epithelial cell lines, but had no effect on growth of Dunning stromal cells or primary SV mesenchyme. Furthermore, FGF10 protein stimulated the development of ventral prostate and seminal vesicle organ rudiments in serum-free organ culture. When both FGF10 and testosterone were added to organs in vitro, there was no synergistic induction of development. Additionally, development induced by FGF10 was not inhibited by the addition of the anti-androgen Cyproterone Acetate demonstrating that the effects of FGF10 were not mediated by the androgen receptor. Taken together, our experiments suggest that FGF10 functions as a mesenchymal paracrine regulator of epithelial growth in the prostate and seminal vesicle and that the FGF10 gene is not regulated by androgens

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

1998 ◽  
Vol 78 (4) ◽  
pp. 587-597 ◽  
Author(s):  
M. Y. Yang ◽  
R. Rajamahendran
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture System ◽  
Physiological Role ◽  
Serum Free ◽  
Igf I ◽  
The Media ◽  
Estradiol 17Β ◽  
Insulin Like Growth Factors

The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5–8 and >8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 105 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P4) and estradiol-17β (E2) in the media were determined by radioimmunoassay. Basal E2 production by GC from follicles of all sizes decreased with time of culture (P < 0.01) while basal P4 production increased (P < 0.01). Basal E2 and P4 production increased with increasing size of follicles (P < 0.01). Only very low concentrations of FSH stimulated E2 production from medium and large follicles. Follicle-stimulating hormone stimulated P4 production by GC of follicles of all sizes (P < 0.05). Luteinizing hormone inhibited E2 production by GC in medium and large follicles (P < 0.05), suggesting that LH is responsible for the rise in plasma E2 through effects on both theca cells and GC. A dose of 100 ng mL−1 of IGF-I increased E2 production by GC from medium and large follicles (P < 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P < 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P < 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. Key words: Granulosa cells, gonadotropins, Insulin-like growth factors, progesterone, estradiol-17β, cows

Mitogenic actions of endothelin and other growth factors in ovine endometrium

1997 ◽  
Vol 152 (2) ◽  
pp. 283-290 ◽  
Author(s):  
L A Salamonsen ◽  
R J Young ◽  
S Garcia ◽  
J K Findlay
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Stromal Cells ◽  
Cellular Proliferation ◽  
Calf Serum ◽  
Dose Dependence ◽  
Leukaemia Inhibitory Factor ◽  
Serum Free

Abstract Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 ± 820 c.p.m./well) and stromal (6368 ± 1350 c.p.m./well) cells and was displaced by ET-1 (1 μmol l−1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l−1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 ± 13% of control (untreated=100%) with dose-dependence between the range of 1 to 100 nmol l−1. Stimulation by fetal calf serum was to 377 ± 126% of control. The effects on proliferation by other growth factors (dose; % of control ± s.e.m., number of positives/total number of cell preparations) were: IGF-I (13 nmol l−1; 182 ± 14, 4/4), epidermal growth factor (EGF; 4·8 nmol l−1; 132 ± 5%, 7/7), platelet-derived growth factor-BB (0·4 nmol l−1; 146 ± 3, 2/2) and leukaemia inhibitory factor (0·4 nmol l−1; 110 ± 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 ± 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation. Journal of Endocrinology (1997) 152, 283–290

Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture

1989 ◽  
Vol 67 (2-3) ◽  
pp. 121-127 ◽  
Author(s):  
Lyne Bertrand ◽  
Normand Brière
Keyword(s):  
Organ Culture ◽  
Brush Border ◽  
In Vitro Maturation ◽  
Kidney Development ◽  
Thymidine Incorporation ◽  
Brush Border Enzymes ◽  
Serum Free ◽  
Foetal Kidney ◽  
Developing Kidney

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13–18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 °C in a mixture of 95% air – 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished γ-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.Key words: hydrocortisone, organ culture, human foetus, kidney, development.

Megakaryocytes Cultured from Patients with IMF Produced Significantly Higher mRNA but Not Protein Levels of Fibrosing Grown Factors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3607-3607
Author(s):  
Jen-Chin Wang ◽  
Tsong H Chang ◽  
Amit Goldberg ◽  
Allan D. Novetsky ◽  
Steven Lichter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Growth Factor ◽  
Mrna Levels ◽  
Serum Free Medium ◽  
Free Medium ◽  
Myeloid Metaplasia ◽  
Serum Free ◽  
Protein Levels

Abstract Currently, the prevailing concept concerning the etiology of bone marrow fibrosis in patients with idiopathic myelofibrosis (IMF) is that it results from excessive production of fibrosing growth factors including transforming growth factor beta (TGF-B1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) from megakaryocytes and monocytes. Since megakaryocytes are difficult to isolate from bone marrow in IMF patients, this concept remains speculative. We obtained megakaryocytes (CD41+ cells) from 10-day in vitro culture of blood CD34+ cells in serum-free medium with thrombopoietin and stem cell factors as described and cultured monocytes from isolating blood CD14+ cells. Then quantitative analyses of fibrosing growth factors at the mRNA and protein levels were obtained. mRNA levels were obtained from real-time RT-PCR technique, and protein levels were obtained from ELISA analysis of the supernatant of CD41+ cells cultured 4 h in serum-free medium. The results showed 1) mRNA levels of TGF-B1, PDGF, and FGF produced by the megakaryocytes were significantly elevated in agnogenic myeloid metaplasia (AMM) compared with those in normal controls (p<0.05). While these growth factors were elevated several-fold in AMM compared with other myeloproliferative disorders (MPD) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) mRNA levels of TGF-B1 were higher than levels of PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r=0.73) but not with monocytes (r=0.23). 5) ELISA analysis of the growth factors from the cultured megakaryocytes showed that, in most of the patients with AMM and other MPDs and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM had significantly elevated protein levels of these growth factors. We conclude thatin IMF, megakaryocytes but not monocytes are the predominant cells producing fibrosing growth factors, andthe failure of finding increased protein levels of these growth factors in the in vitro system suggest that other factors are necessary to initiate translation of these growth factors in the megakaryoctes, and neutrophil emperipolesis with releasing factors may be important in this process.

A serum-free in vitro model of renal microvessel development

1998 ◽  
Vol 274 (6) ◽  
pp. F1150-F1160 ◽  
Author(s):  
Lisa M. Antes ◽  
Monica M. Villar ◽  
Sylvia Decker ◽  
Roberto F. Nicosia ◽  
Dean A. Kujubu
Keyword(s):  
Growth Factors ◽  
Endothelial Cell ◽  
In Vitro Model ◽  
Type I Collagen ◽  
Type I ◽  
Renal Vasculature ◽  
Serum Free ◽  
Polypeptide Growth Factors ◽  
Vitro Model

The differentiation and organization of the embryonic renal vasculature is a crucial event in renal development. To study this process, we developed a serum-free in vitro model of renal microvessel development. Mouse embryonic kidney explants, when embedded specifically in type I collagen, demonstrate outgrowth of microvascular structures when stimulated by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA, 10–50 ng/ml). Other polypeptide growth factors stimulated little, if any, microvessel outgrowth from the explants. Similar outgrowths were not observed when other embryonic tissue explants were used. The number of microvessels observed depended on the gestational age of the explants. We hypothesize that TPA induces the in situ differentiation of metanephric mesenchymal cells into endothelial cell precursors and that specific matrix proteins and cell-matrix interactions are necessary for the organization of these precursors into microvessels. Our model will allow us to examine in detail the responsiveness of metanephric kidney cells to both growth factors and extracellular matrix molecules and to understand how they influence renal endothelial cell differentiation.

Sign in / Sign up

Export Citation Format

Share Document