Erythropoietin-dependent endothelial proteins: potential use against erythropoietin resistance.

The endothelium was the first non-hematopoietic tissue to be identified as a physiological target for erythropoietin (EPO). EPO is involved in recruitment and mobilization of endothelial progenitors and stimulates the production of erythroid cell regulatory factors in endothelial cells. Production of these EPO-dependent factors is inhibited by IL-3 in vitro.

Biological activities and molecular interactions of the C-terminal residue of thrombospondin-4, an epitome of acidic amphipathic peptides.

C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid stimulating factors of endothelial origin. It is active in cultures of several human hematopoietic stem cells, skin fibroblasts and kidney epithelial cells and stimulates red cell formation in anemic mice. A method of affinity chromatography in the presence of high concentrations of Triton X-100, previously developed for identifying proteins associated with the TSP-1 receptor CD47, was utilized for the detection of C21 binding molecules and their detergent-resistant, associated partners.

Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation.

Background: C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator.

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin.

Chaperonin 10 as an endothelial-derived differentiation factor: role of glycogen synthase kinase-3.

The anti-inflammatory peptide early pregnancy factor/chaperonin 10 (cpn10) was identified by 2D-electrophoresis/mass spectrometry as one of the proteins increased in human umbilical cord endothelial cells (HUVEC) after treatment with erythropoietin (EPO). EPO increased the amount of cpn10 released into the medium of HUVEC cultures, despite the absence of a secretory signal peptide. Although immunosupressive agents would represent an indirect advantage for red cell formation under conditions of infection and inflammation, it is possible that cpn10 may have direct effects on erythroid cells.

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing.

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts.

Serpin A1 and CD91 as host instruments against HIV-1 infection: are extracellular antiviral peptides acting as intracellular messengers?

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor) has been shown to be a non-cytolytic antiviral factor present in blood and effective against HIV infection. The best known physiological role of serpin A1 is to inhibit neutrophil elastase, a proteinase which is secreted by neutrophils at sites of infection and inflammation. Decreased HIV-infectivity is associated with decreased density of membrane-associated elastase.

The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1.

Serpin A1 (alpha1-proteinase inhibitor) inhibits human immunodeficiency virus 1 (HIV-1) production by mechanisms which remain to be elucidated. The complex formation of serpin A1 with proteinases eliminates the proteolytic activity and generates a fragment corresponding to the serpin C-terminal 36-residue peptide. Here, we show that the C-terminal 26-residue peptide of serpin A1 (A1-C26) inhibits HIV-long terminal repeat (LTR)-driven transcription in epithelial cells transfected with HIV-1 LTR promoter-driven genes.

Monitoring insulin-like growth factors in HIV infection and AIDS.

There is a close association between the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, infection and immunity. Infection with the human immunodeficiency virus (HIV) is often associated with a decrease of the concentrations of IGF-I, IGF-II, IGF-binding protein 3 (IGFBP-3) and an increase of IGFBP-1 and -2. Many investigators have studied the relationship between the GH-IGF-I system and some of the most common characteristics of disease progression, such as decreased CD4 cell counts, weight loss and fat redistribution.

Thrombospondin 1, produced by endothelial cells under the action of erythropoietin, stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of insulin-like growth factor binding protein 3.

The nature of erythropoietin (EPO)-dependent, erythroid cell regulatory factors secreted by endothelial cells is largely unknown. The production of thrombospondin 1 (TSP-1) and insulin-like growth factor binding protein 3 (IGFBP-3) is increased in cultures of human umbilical vein endothelial cells (HUVEC) incubated with erythropoietin (EPO). Simultaneous incubation of HUVEC with EPO and interleukin 3 (IL-3) resulted in a decreased production, suggesting that both TSP-1 and IGFBP-3 belong to the EPO- and IL-3-dependent erythroid regulatory factors previously described in cultures of bone marrow endothelial cells.

The C-terminal 26-residue peptide of serpin A1 stimulates proliferation of breast and liver cancer cells: role of protein kinase C and CD47.

C26, the C-terminal 26 residue peptide of serpin A1, significantly increased cell proliferation in cultures of hepatoma cells, but not in porcine kidney epithelial cells, human skin fibroblasts or keratinocytes. The mitogenic activity of C26 was preferentially inhibited with a protein kinase C (PKC) inhibitor, an antibody against CD47 and CD47 short interfering RNA. The mutant C26-K19R,N22M, imitating a thrombospondin-like cell adhesion motif, increased the mitogenic activity in both Hep G2 cells and MCF-7 breast cancer cells.

The C-terminal peptide of thrombospondin-4 stimulates erythroid cell proliferation.

Erythropoietin (EPO) stimulates the production of small erythroid cell stimulating factors (molecular weight <5 kDa) in cultures of bone marrow endothelial cells. We identified a fragment of thrombospondin-4 (TSP-4) as an EPO-stimulated protein in endothelial cell lysates. Pre-incubation of the low molecular weight fractions from supernatants of EPO-treated umbilical cord endothelial cells (HUVEC) with antibodies against the C-terminal residues of TSP-1,2 and TSP-4 decreased the erythroid cell stimulating activity.

The fusion of IGF I with stromal cell-derived factor I or alpha1 proteinase inhibitor alters their mitogenic or chemotactic activities while keeping their ability to inhibit HIV-1-gp120 binding.

It has been previously reported that insulin-like growth factor I (IGF I) decreases in AIDS patients with wasting, a condition that is partially prevented by combined IGF I growth hormone therapy. By generating bifunctional proteins of IGF I and stromal cell-derived factor 1alpha (SDF-1alpha) or alpha1 proteinase inhibitor (API), two proteins known to prevent HIV infection, it may be possible to improve the therapeutic effectiveness of these compounds for the treatment of AIDS-mediated wasting. SDF-1alpha or the M351E-M358L mutant of API were attached at the C-terminal end of IGF I and synthesized by a stable insect cell expression technique.

The improved survival of hematopoietic cells cultured with a fusion protein of insulin-like growth factor II (IGF-II) and interleukin 3 (IL-3) is associated with increases in Bcl-xL and phosphatidylinositol-3 kinase activity.

We compared the antiapoptotic activity of a recombinant chimera of insulin-like growth factor II (IGF-II) and interleukin (IL)-3 with the corresponding equimolar mixture of the individual components based on changes in several factors associated with survival in the CD34+ human hematopoietic cell line TF-1. Propidium iodide-stained cells analyzed by fluorescein-activated cell sorter indicated that the chimera was more effective than the corresponding equimolar mixture in decreasing the amounts of apoptotic cells and increasing the proportion of cells in the S-phase of the cell cycle. The chimera was more effective in increasing the antiapoptotic protein Bclx(L) and produced a significant increase in signal transducer and activator of transcription-5 phosphorylation and in phosphatidylinositol-3 kinase (PI-3K) activity.

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells.

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells.

Enhanced proliferative effects of a baculovirus-produced fusion protein of insulin-like growth factor and alpha(1)-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351.

Alpha(1)-proteinase inhibitor (API) was coupled at the C-terminus of a human insulin-like growth factor (IGF) analog to facilitate its production in insect cells. This fusion protein significantly increased thymidine incorporation into HL-60 cells as compared with the incorporation observed with an equivalent molar mixture of the IGF analog and API. The M351E variant of API has been previously shown to reduce aggregate formation in prokaryotic expression systems.

Insect cell production of a secreted form of human alpha(1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities.

alpha(1)-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells.

Efficacy of an insulin-like growth factor-interleukin-3 fusion protein in reversing the hematopoietic toxicity associated with azidothymidine in mice.

The effect of 406, a novel fusion protein between the N-terminal sequence of the insect insulin-like peptide, bombyxin, human insulin-like growth factor II and mouse interleukin 3 was investigated in its capacity to abrogate the toxic effects of azidothymidine (AZT) in C57BL/6 mice. Mice receiving 2.5 mg/ml AZT in their drinking water were concurrently treated with daily s.

Preparation of a recombinant chimaera of insulin-like growth factor II and interleukin 3 with high proliferative potency for haemopoietic cells.

We have found that a slightly modified insulin-like growth factor II (IGF II) consisting of a chimaera of bombyxin and human IGF II (BOMIGF) is properly secreted in insect cells by using the baculovirus expression system. Human interleukin 3 (IL-3) was attached to the C-terminal amino acid residue of BOMIGF with peptide linkers containing five or twelve residues. Only the chimaera with the 12-residue linker had biological activities of both IGF II and IL-3.

The influence of various insect cell lines, p10 and polyhedrin promoters in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system.

A technique for the optimal synthesis of secreted fusion proteins between insulin-like growth factors (IGFs) and cytokines is described. The cDNA of BOMIGF, a fusion protein between the insect insulin-like peptide bombyxin and IGF II, has been linked to the gene of interleukin-3. The BOMIGF-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer vectors.

Increased heparin binding by site directed mutagenesis of a recombinant chimera of bombyxin and insulin-like growth factor II.

BOMIGF, a chimera between bombyxin and insulin-like growth factor II (IGF II) has been modified and extended to a total of 85 amino acids including 7 additional basic amino acids. The new peptide bound heparin as efficiently as GM-CSF and much better than IGF II or BOMIGF. It could be retained on albumin-coated tissue culture wells without loosing its capacity to stimulate thymidine incorporation into erythroid cells of fetal bovine liver and on fibronectin-heparin coated wells keeping its mitogenic activity towards L6 muscle cells.

Bovine fetal-liver stromal cells support erythroid colony formation: enhancement by insulin-like growth factor II.

Stromal cells are one of the components of the hematopoietic microenvironment, which is crucial for the proliferation and differentiation of hematopoietic cells. We have obtained a bovine fetal-liver stromal cell line that supports erythroid colony formation in the presence of erythropoietin. The cells are cytokeratin-negative and vimentin-positive, indicating a mesenchymal origin.

Accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor II and an insect insulin-like peptide.

A synthetic gene encoding the signal peptide and the N-terminal sequence of bombyxin, an insect insulin-like peptide, and the 58 amino acids of the C-terminal sequence of human insulin-like growth factor II (IGF II) has been expressed using the baculovirus system. This synthetic chimaera was obtained by amplification of four overlapping oligonucleotides using Taq polymerase and cloning into the transfer vector pBluebac. The construct was integrated by homologous recombination into the Autographa californica nuclear polyhedrosis genome.