Biochemical characterization of bona fide polycystin-1 in vitro and in vivo.

The most common form of autosomal dominant polycystic kidney disease (PKD) results from mutation of the PKD1 gene on chromosome 16p13.3. The gene encodes a 14-kb messenger RNA that is predicted to express a 462-kd membrane protein. The gene product, polycystin-1, has a large extracellular portion composed of a novel combination of protein-protein interacting domains and is postulated to be a plasma membrane receptor involved in cell-cell/matrix interactions. However, slow progress has been made in the characterization of polycystin-1 or the determination of its function. In fact, the protein is expressed at very low levels in tissues and cell lines and previous efforts directed at expression of recombinant protein had been largely unsuccessful. We have recently developed constructs of full-length human PKD1 complementary (cDNA) that can be expressed in both a stable and transient fashion in mammalian cells. We used these systems to characterize our antibodies and to track the protein in vivo. We report here the first biochemical characterization of recombinant polycystin-1 and show that the protein is a 520-kd glycosylated polypeptide with an unglycosylated core of 460 kd. Subcellular fractionation as well as biotinylation studies confirmed that the protein is plasma-membrane associated. Furthermore, we show that the recombinant protein localizes to cell-cell junctions in polarized madin darby canine kidney cells as revealed by indirect immunofluorescence. Our data represent the first characterization of polycystin-1 performed under highly controlled conditions.

Response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: Relevance to the regulation of angiogenesis.

Angiogenesis, the formation of new capillary blood vessels, occurs almost exclusively in the microcirculation. This process is controlled by the interaction between factors with positive and negative regulatory activity. In this study, we have compared the effect of two well described positive regulators, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) on bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. The parameters we assessed included (a) cellular reorganization and lumen formation following exposure of the apical cell surface to a three-dimensional collagen gel; (b) organization of the actin cytoskeleton; (c) expression of thrombospondin-1 (TSP-1), an endogenous negative regulator of angiogenesis; and (d) extracellular proteolytic activity mediated by the plasminogen activator (PA)/plasmin system. We found that (a) collagen gel overlay induces rapid reorganization and lumen formation in BME but not BAE cells; (b) FGF-2 but not VEGF induced dramatic reorganization of actin microfilaments in BME cells, with neither cytokine affecting BAE cells; (c) FGF-2 decreased TSP-1 protein and mRNA expression in BME cells, an effect which was specific for FGF-2 and BME cells, since TSP-1 protein levels were unaffected by VEGF in BME cells, or by FGF-2 or VEGF in BAE cells; (d) FGF-2 induced urokinase-type PA (uPA) in BME and BAE cells, while VEGF induced uPA and tissue-type PA in BME cells with no effect on BAE cells. Taken together, these findings reveal endothelial cell-type specific responses to FGF-2 and VEGF, and point to the greater specificity of these cytokines for endothelial cells of the microvasculature than for large vessel (aortic) endothelial cells. Furthermore, when viewed in the context of our previous observation on the synergistic interaction between VEGF and FGF-2, our present findings provide evidence for complementary mechanisms which, when acting in concert, might account for the synergistic effect.

Characterization of novel clonal murine endothelial cell lines with an extended life span.

A murine endothelial cell line was recently established from microvessels that had invaded a subcutaneous sponge implant (Dong, Q. G.; Bernasconi, S.; Lostaglio, S., et al. Arterioscl. Thromb. Vasc. Biol. 17:1599-1604; 1997). From these sponge-induced endothelial (SIE) cells, we have isolated two subpopulations endowed with different phenotypic properties. Clone SIE-F consists of large, highly spread cells that have a relatively slow growth rate, form contact-inhibited monolayers, do not grow under anchorage-independent conditions, express elevated levels of thrombospondin-1 (TSP-1) and are not tumorigenic in vivo. In contrast, clone SIE-S2 consists of small, spindle-shaped cells that have a high proliferation rate, do not show contact-inhibition, grow under anchorage-independent conditions, express very low levels of TSP-1 and are tumorigenic in vivo. Both clones express the endothelial markers vascular endothelial-cadherin and vascular intercellular adhesion molecule-1, but do not express CD31 and E-selectin. In addition, SIE-S2 cells, but not SIE-F cells, express the alpha-smooth muscle actin isoform. SIE-S2 cells, but not SIE-F cells, are able to form branching tubes in fibrin gels. The SIE-F and SIE-S2 clones, which have properties of nontransformed and transformed cells, respectively, should provide useful tools to investigate physiological and pathological processes involving vascular endothelium.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins.

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

Glomerular filtration is required for transfection of proximal tubular cells in the rat kidney following injection of DNA complexes into the renal artery.

Gene transfer to the kidney can be achieved with various DNA vectors, resulting in transgene expression in glomerular or tubular districts. Controlling transgene destination is desirable for targeting defined renal cells for specific therapeutic purposes. We previously showed that injection of polyplexes into the rat renal artery resulted in transfection of proximal tubular cells. To investigate whether this process involves glomerular filtration of the DNA-containing particles, fluorescent polyethylenimine polyplexes were prepared, containing fluoresceinated poly-L-lysine. This allowed visualization of the route of the particles into the kidney. Our polyplexes were filtered through the glomerulus, since fluorescent proximal tubuli were observed. Conversely, fluorescent lipopolyplexes containing the cationic lipid DOTAP were never observed in tubular cells. Size measurements by laser light scattering showed that the mean diameter of polyplexes (93 nm) was smaller than that of lipopolyplexes (160 nm). The size of the transfecting particles is therefore a key parameter in this process, as expected by the constraints imposed by the glomerular filtration barrier. This information is relevant, in view of modulating the physico-chemical properties of DNA complexes for optimal transgene expression in tubular cells. Gene Therapy (2000) 7, 279-285.

Cytosolic immunization allows the expression of preATF-saporin chimeric toxin in eukaryotic cells.

In this work, we have devised an intracellular immunization strategy for the expression in high amounts of ATF-saporin, a targeted chimeric toxin constituted by the ATF receptor binding domain of human urokinase and the plant ribosome-inactivating protein saporin, which has been shown to be highly cytotoxic to target cells. This strategy may allow the production of highly toxic secretory proteins in eukaryotic cells, avoiding cell suicide caused by autointoxication. The procedure consists of equipping host cells with cytosolic neutralizing antibodies directed toward the toxic domain of the heterologous polypeptide. We show that this intracellular immunization is essential for the synthesis of correctly folded, biologically active ATF-SAP in the high amounts needed to investigate its in vivo anti-metastatic potential. Such a strategy should be generally useful for the production of toxic molecules of therapeutic value whose folding and maturation require transit through the eukaryotic secretory pathway. Fabbrini, M. S., Carpani, D., Soria, M. R., Ceriotti, A. Cytosolic immunization allows the expression of preATF-saporin chimeric toxin in eukaryotic cells.

Phenotypic alterations in senescent large-vessel and microvascular endothelial cells.

Endothelial cell senescence likely plays a key role in age-associated vascular diseases. A close relationship between in vitro and in vivo senescence of endothelial cells has been established. Therefore, elucidating the structural and functional changes occurring during long-term cultures of endothelial cells would contribute to clarifying the pathogenesis of vascular disorders in the elderly. We investigated the effects of replicative senescence on the architecture of bovine aortic vs microvascular endothelial cells. A marked increase in cell area was observed in both cell types, whereas dramatic morphological alterations were detected in microvascular endothelial cells only. The latter also showed age-associated reorganization of the actin cytoskeleton. Finally, both aortic and microvascular endothelial cells lost their migratory response to basic fibroblast growth factor with age. Our results highlight dramatic structural and functional alterations in senescent endothelial cells. Such rearrangements might account for in vivo endothelial cell alterations involved in age-associated vascular dysfunction.

Exogenous fibroblast growth factor-2 induces a transformed phenotype in vascular kaposi's sarcoma-like cells.

Vascular TTB cells derive from murine Kaposi's sarcoma-like dermal lesions and share several phenotypic features with AIDS-associated KS spindle cells. We have recently reported that fibroblast growth factor-2 (FGF-2) promotes dramatic cytoskeletal and morphological alterations in TTB cells, concomitant with the induction of an autocrine loop for hepatocyte growth factor and a relocalization of the urokinase receptor. Since all these alterations are hallmarks of cell transformation. we attempted to verify whether FGF-2 induces a transformed phenotype in TTB cells. Our results show that FGF-2-treated TTB cells acquire the ability to grow under anchorage-independent conditions. In addition, FGF-2 markedly reduced the levels of thrombospondin-1, an antiangiogenic and tumor suppressor protein, in TTB cells. Therefore, FGF-2 induces KS-like spindle cells to acquire properties characteristic of transformed cells. This suggests that FGF-2 plays a pathogenetic role in KS not only by promoting angiogenesis, but also by conferring a transformed phenotype upon KS cells. In light of previous reports on Tat-induced release of FGF-2 into the extracellular space, our findings may provide an additional mechanism for the observed synergism between Tat and FGF-2 in the pathogenesis of KS.

EDF-1, a novel gene product down-regulated in human endothelial cell differentiation.

Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encoding EDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridging factor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiation in vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation.

FGF-2 stimulates migration of Kaposi's sarcoma-like vascular cells by HGF-dependent relocalization of the urokinase receptor.

The spindle-shaped cell line TTB was recently isolated from highly vascularized skin lesions of BKV/HIV-1 tat transgenic mice and shown to possess an autocrine loop for hepatocyte growth factor (HGF). We show that fibroblast growth factor-2 (FGF-2) stimulates TTB cell migration and promotes polarization of uPAR at the leading edge of migrating cells. FGF-stimulated TTB cells presented the typical migratory phenotype, with a triangular cell shape and concomitant breakdown of actin stress fibers and smooth muscle-specific actin isoform. FGF-2-stimulated migration was blocked by antibodies against urokinase-type plasminogen activator (uPA) or uPA receptor (uPAR) and by neutralizing anti-HGF antibodies. The latter also inhibited uPAR relocalization at the cell surface of FGF-2-treated TTB cells. This points to a crosstalk between FGF-2 and HGF that might mediate TTB cell migration by modulating the localization of cell surface uPAR.

Fibronectin modulates the effects of HIV-1 Tat on the growth of murine Kaposi's sarcoma-like cells through the down-regulation of tyrosine phosphorylation.

HIV-1 Tat plays a role in the pathogenesis of Kaposi's sarcoma. We therefore investigated the effect of Tat on the growth of murine Kaposi's sarcoma-like spindle (TTB) cells derived from dermal lesions. We observed that Tat and a peptide corresponding to the carboxyl-terminal region (Tat65-80) containing an RGD sequence inhibit TTB cell proliferation only when cells are cultured on fibronectin. This inhibitory effect correlates with redistribution of the alpha(v) integrin subunit on the surface of TTB cells and with down-regulation of tyrosine phosphorylation of specific substrates due to an increased tyrosine phosphatase activity. Indeed, phenylarsine oxide, a potent inhibitor of phosphotyrosine phosphatases, prevented the effects of Tat on TTB cells. We therefore argue that the action of Tat on TTB cells is mediated by the RGD motif through an integrin-based cell signaling pathway involving the activity of phosphotyrosine phosphatase(s), which would lead to a decrease in the levels of phosphotyrosine-containing proteins, among which is erk-2/p42MAPK.

The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated.

In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.

Fibronectin modulates endothelial response to HIV type 1 Tat.

The normal function of the endothelium is impaired in HIV-1 infection. Disturbances of the local cytokines as well as the release of HIV-1 Tat by infected mononuclear cells play a role in endothelial dysfunction. We studied the effects of Tat on the human endothelial ECV cell line. In this system, Tat inhibited cell proliferation only in the presence of fibronectin as a culture substrate, whereas it did not modulate plasminogen activator activity, cell migration, or synthesis of fibronectin. Because amino acids 49-57 contains a nuclear translocation sequence, we also evaluated the potential intracellular role of Tat in tat-transfected ECV cells. tat transfectants showed inhibition of cell growth, unaffected cell migration and plasminogen activator activity, and a significant induction of the expression of fibronectin.

Nanoscopic structure of DNA condensed for gene delivery.

Scanning force microscopy was used to examine DNA condensates prepared with varying stoichiometries of lipospermine or polyethylenimine in physiological solution. For the first time, individual DNA strands were clearly visualized in incomplete condensates without drying. Using lipospermine at sub-saturating concentrations, discrete nuclei of condensation were observed often surrounded by folded loops of DNA. Similar packing of DNA loops occurred for polyethylenimine-induced condensation. Increasing the amount of the condensing agent led to the progressive coalescence or aggregation of initial condensation nuclei through folding rather than winding the DNA. At over-saturating charge ratios of the cationic lipid or polymer to DNA, condensates had sizes smaller than or equal to those measured previously in electron micrographs. Polyethylenimine condensates were more compact than lipospermine condensates and both produced more homogeneously compacted plasmids when used in a 2-4-fold charge excess. The size and morphology of the condensates may affect their efficiency in transfection.

Nonviral gene delivery to the rat kidney with polyethylenimine.

The aim of this study was to establish a nonviral method for gene delivery to the rat kidney. To this purpose, a panel of reagents was tested, including a monocationic lipid, DOTAP, a polycationic lipid, DOGS (or Transfectam), and three different forms of the cationic polymer polyethylenimine (PEI). A comparison among these compounds was performed in vivo, using luciferase as reporter gene. Complexes containing 10 microg of DNA were injected into the left renal artery of rats and allowed to remain in contact with the kidney for 10 min. Forty-eight hours later, luciferase expression levels in kidney extracts were measured. Kidneys injected with DNA complexed to the branched, 25-kD PEI polymer (PEI 25k) yielded activity levels significantly higher than control, sham-operated kidneys (2.7 x 10(4) vs. 5.2 x 10(3) RLU/kidney, respectively), whereas the other transfecting agents did not yield significant activity over controls. PEI 25k was therefore chosen for further optimization of transfection conditions. Dose-dependent luciferase expression was shown for 10, 50, and 100 microg of PEI-complexed DNA, reaching maximal levels of 2.4 x 10(5) RLU/kidney at 100 microg DNA. The optimal PEI nitrogen/DNA phosphate molar ratio was 10 equivalents. Luciferase activity peaked at 2 days, was still significantly higher than controls at 7 days, and was undetectable at 14 days post-injection. Using beta-galactosidase (beta-Gal) as a reporter, transgene expression was localized almost exclusively in proximal tubular cells.

Characterization of a saporin isoform with lower ribosome-inhibiting activity.

We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands.

Expression of recombinant human granulocyte colony-stimulating factor in CHO dhfr- cells: new insights into the in vitro amplification expression system.

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.

[Prevalence of dental caries in a 6-year old population from San Fernando (Cádiz)]. / Prevalencia de la caries dental en la población de 6 años de San Fernando (Cádiz).

AIM: The aim of this survey was to know the dental caries prevalence and severity in the 6 years old population from San Fernando (Cádiz). DESIGN: This is a cross sectional survey. EMPLACEMENT: We studied every schoolchildren at this age (100%) from the 27 city schools, in 1993. PARTICIPANTS: We studied 1439 schoolchildren of this age. This is one of the ages recommended by WHO in this class of surveys. INTERVENTIONS: We made an oral exploration following the WHO recommendations in this type of surveys. RESULTS: Dental caries prevalence reached 79% at this age. Dental decay index were; df-t index was 3.19; DMF-T was 0.68 and filling index was 11.2%. CONCLUSIONS: Our results of caries prevalence and epidemiological dental index, are higher in comparison with the results of other studies, which shows the severity of the dental decay problem in San Fernando. We still need educational and preventive programs in order to decrease dental caries prevalence and severity.