Complications related to fracture treatment in HIV patients: a case report.

We present the case report of a 40-year-old woman who was HIV-positive in Highly Active Anti-Retroviral Therapy (HAART) and affected by femural pertrochanteric fracture, which was treated by endomedullary nailing. Two years after the surgical operation, the woman developed an aseptic symptomatic osteolysis around the implant. Hardware removal was resolutive. Aseptic and septic hardware mobilization, hardware removal, and implant decision in HIV patients with pertrochanteric fractures is discussed. The authors suggest close follow-up and prompt hardware removal, as soon as X-rays demonstrate healing signs, in HIV patients with fracture fixation, if general condition allows.

The crystal structure of human placenta growth factor-1 (PlGF-1), an angiogenic protein, at 2.0 A resolution.

The angiogenic molecule placenta growth factor (PlGF) is a member of the cysteine-knot family of growth factors. In this study, a mature isoform of the human PlGF protein, PlGF-1, was crystallized as a homodimer in the crystallographic asymmetric unit, and its crystal structure was elucidated at 2.0 A resolution. The overall structure of PlGF-1 is similar to that of vascular endothelial growth factor (VEGF) with which it shares 42% amino acid sequence identity. Based on structural and biochemical data, we have mapped several important residues on the PlGF-1 molecule that are involved in recognition of the fms-like tyrosine kinase receptor (Flt-1, also known as VEGFR-1). We propose a model for the association of PlGF-1 and Flt-1 domain 2 with precise shape complementarity, consider the relevance of this assembly for PlGF-1 signal transduction, and provide a structural basis for altered specificity of this molecule.

Effect of placenta growth factor-1 on proliferation and release of nitric oxide, cyclic AMP and cyclic GMP in human epithelial cells expressing the FLT-1 receptor.

We investigated the effect of placenta growth factor-1 (P1GF-1) on cell growth and on the release of nitric oxide (NO), cyclic AMP (cAMP) and cyclic GMP (cGMP) in human malignant epithelial cells. A noteworthy increase in proliferation was induced in choriocarcinoma cells (BeWo) by P1GF-1 treatment, while breast cancer cells (CG-5) were minimally affected. Western blotting and immunocytochemistry demonstrated the expression of the P1GF-1 receptor fms-like tyrosine kinase-1 (Flt-1) in these models. NO was released in the BeWo culture medium as a result of P1GF-1 treatment, with maximal induction occurring after 6 h. Enhanced cAMP levels were observed after 80 min-6 h, while the amounts of cGMP produced were undetectable. In summary, PIGF-1 stimulates the proliferation of cell types that express Flt-1, other than endothelial cells. In BeWo cells, this effect is preceded by the induction of NO and cAMP as probable downstream effectors of Flt-1 activation.

Recombinant production of PIGF-1 and its activity in animal models.

In this paper we review current knowledge on placenta growth factor (PIGF) and summarise our data on its recombinant production in bacteria and its activity. PIGF and vascular endothelial growth factor (VEGF) are both angiogenic factors belonging to the platelet-derived growth factor (PDGF) family. PIGF is a dimeric glycoprotein which shares a number of biochemical and functional features with VEGF. The aminoacidic similarity between the two factors is high (about 50%) in the PDGF-like domain. By alternative splicing of the PIGF mRNA, three forms of PIGF protein are generated which are named PIGF-1, PIGF-2 and PIGF-3. We have focused our attention on form 1 of human PIGF (PIGF-1). A large quantity of active recombinant PIGF-1 has been obtained using a bacterial expression system. By optimising the fermentation and purification it was possible to produce about 140 mg/l of culture of active PIGF-1, which is potentially suitable for a pharmaceutical use. The angiogenic activity of two different batches of bacteria-derived PIGF-1 obtained in our laboratory was demonstrated in chick chorionallantoic membrane assays. Finally, in preliminary studies we have shown that bacteria-derived PIGF-1 has a protective effect against myocardial lesions induced by isoprenaline in rat and rabbit.

Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic.

The placental-derived growth factor (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial growth factor. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic 21-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this factor functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial growth factor and basic fibroblast growth factor under the same conditions and at the same concentration. PIGF-1 was shown to induce cell growth and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial growth factor and basic fibroblast growth factor on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

The placenta growth factor gene of the mouse.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are angiogenic factors containing the 8-cysteine motif of platelet-derived growth factor (PDGF). Both PlGF and VEGF are mitogens for endothelial cells in vitro and promote neoangiogenesis in vivo. In addition, PlGF strongly potentiates the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. We have now isolated the cDNA coding for mouse Plgf by screening a mouse heart cDNA library with the human PlGF sequence as probe. The human PlGF protein has two forms, PlGF-1 and PlGF-2, that arise from alternative splicing of a single gene mapping on Chromosome (Chr) 14; the isolated mouse Plgf cDNA encodes the longer of these two forms (PlGF-2). We show that the mouse Plgf-2 mRNA is the only transcript present in the normal tissues analyzed. Mouse Plgf-2 is a 158-amino-acid-long protein that shows 78% similarity (65% identity) to the human PlGF-2. Computer analysis reveals a putative signal peptide and three probable N-glycosylation sites, two of which are also conserved in human PlGF. The mouse Plgf gene was isolated and characterized; the gene is encoded by 7 exons spanning a 13-kb DNA interval. Finally, we have mapped the mouse Plgf gene to Chr 12, one cM from D12Mit5, and the human PlGF gene to 14q24, using both FISH and genetic crosses.

Upregulation of vascular endothelial growth factor (VEGF) and downregulation of placenta growth factor (PlGF) associated with malignancy in human thyroid tumors and cell lines.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells in vitro, promotes neoangiogenesis in vivo and increases the permeability of the vascular endothelium. VEGF overexpression occurs in several cultured tumor cell lines and in certain human malignancies. Placenta growth factor (PlGF) is a recently identified growth factor for endothelial cells (EC); PlGF strongly potentiates both the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. To uncover the molecular mechanisms underlying neoangiogenesis in human thyroid tumors, we have analysed VEGF and PlGF expression in a panel of thyroid carcinoma cell lines with different tumorigenic potential as well as in human primary thyroid tumors. We show that a high tumorigenic potential is associated with an elevated VEGF expression in human thyroid tumor cell lines. Furthermore, VEGF overexpression occurs in 5/5 highly malignant anaplastic carcinomas. Papillary and follicular carcinomas express intermediate levels of VEGF mRNA. In contrast, PlGF expression is severely down regulated in the majority of thyroid tumor cell lines and in tumors. Furthermore, we show that both the VEGF receptors, FLT-1 and flk/KDR, are expressed in endothelial cells that line tumor-embedded microvascular vessels, suggesting that VEGF but not PlGF, contributes to thyroid tumor development.

VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor which binds to two structurally related tyrosine kinase receptors denoted KDR and FLT1. To compare the interaction of VEGF with each receptor, cell lines which express individual receptor subtypes were identified using Northern blot hybridization. Bovine aortic endothelial (ABAE) cells and WM35 melanoma cells were found to express KDR, while FLT1 was primarily expressed on SK-MEL-37. Both receptor subtypes were detected on another melanoma cell line (WM9). Heparin augmented VEGF binding to KDR-expressing cells (ABAE and WM35), but inhibited VEGF binding to FLT1-expressing cells (SK-MEL-37 and WM9). The concentration of heparin required for half maximal stimulation of VEGF binding to KDR-expressing cells (500 ng/ml) was 25 times greater than that required for half maximal inhibition of binding to FLT1-expressing cells (20 ng/ml). In WM9 cells, the effect of heparin was bimodal; low concentration inhibited, while higher concentrations stimulated binding of 125I-VEGF. Placenta growth factor (PIGF-1) is a recently described growth factor structurally similar to VEGF. PIGF-1 had a negligible or no effect on 125I-VEGF binding to KDR-expressing cells (ABAE, WM35), but did complete for binding to FLT1-expressing cells (SK-MEL-37 and WM9). Addition of heparin had no effect on its ability to compete for binding with 125I-VEGF. The data indicate differential regulation of the two VEGF receptors by heparin and extended specificity of FLT1 receptor, but not KDR, for binding PIGF-1 growth factor.

Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.

We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.

Translation of the placenta growth factor mRNA is severely affected by a small open reading frame localized in the 5' untranslated region.

The recently identified placenta growth factor gene (PIGF) code for a protein related to the vascular permeability factor (VPF). We present evidence indicating that expressing of this gene could be regulated at the post-transcriptional level. The region upstream to the coding region of PIGF mRNA contains a small open reading frame (ORF), potentially coding for a peptide of 15 amino acids. The translation of different constructs in reticulocyte and wheat germ lysates as well as in COS-1 and CV-1 cells indicates that this short region is a translational inhibitory element since mutations in its two potential initiator codons increase PIGF synthesis in vivo. Using RNAse protection assay, we demonstrate that the PIGF mRNAs obtained from human term placenta and JEG choriocarcinoma cell line have a complete 5' untranslated region and, consequently, also the above mentioned small ORF. Finally, the analysis of a bovine PIGF genomic clone reveals that this small ORF is strongly conserved with respect to both putative peptide sequences and distance from the PIGF coding region.

Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.

A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.

Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.

Identification of new human repetitive sequences: characterization of the corresponding cDNAs and their expression in embryonal carcinoma cells.

We have identified new repeated interspersed DNA sequences by analysis of homologous RNA transcripts from a human teratocarcinoma cell line (NTERA-2 clone D1). The abundance of transcripts varies upon retinoic acid induced differentiation of NTERA-2/D1 cells, and it is highest when the cells display the embryonal carcinoma phenotype. The expression of these novel repeated sequences appears to be tissue specific as no detectable expression was found in various cell lines of different embryological derivation. Characterization of the RNA transcripts by analysis of recombinant cDNA clones indicated that transcripts of different genomic units are present in undifferentiated embryonal teratocarcinoma cells. Nucleotide sequencing of the cloned cDNAs reveals a complex structure composed by unique and tandemly repeated sub-elements.