Endoplasmic reticulum-resident chaperones modulate the inflammatory and angiogenic responses of endothelial cells.

BACKGROUND: Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. OBJECTIVES: To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. METHODS: Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on their inflammatory response and angiogenic potential. RESULTS: In all chronic wounds investigated, the levels of these ER-resident chaperones were elevated in endothelial cells and leucocytes. The proangiogenic role of BiP has been shown in tumour growth studies before and was confirmed in this study. Proangiogenic activity of the cochaperone Mdg1 has been postulated before but could not be confirmed in this study. The chemokine tumour necrosis factor (TNF)-α was shown to trigger the presentation of proinflammatory adhesion molecules and the release of proinflammatory cytokines. Here we show that TNF-α does not affect endogenous chaperone levels, but that the ER-resident chaperones BiP and Mdg1 modulate the cellular TNF-α-induced proinflammatory response. CONCLUSIONS: According to the presented data we assume that in chronic wounds upregulated levels of ER-resident chaperones might contribute to persistent inflammation in chronic wounds. Therapies to downregulate chaperone levels might provide a tool that switches the imbalanced chronic wound microenvironment from inflammation to healing.

The role of Emx2 during scapula formation.

The scapula is subdivided into head, collum, and blade. Due to the expression pattern of Emx2 and the absence of the scapula blade in Emx2 knockout mice, it has been suggested that Emx2 is involved in the formation of the scapula. Micromanipulation experiments revealed that ectoderm ablation over the somites does not affect Emx2 expression but inhibits the formation of the scapula blade indicating that Emx2 is not sufficient to induce scapula blade formation. Furthermore, we show that the formation of the scapula head is dependent, scapula blade formation independent of FGFR-1-mediated signaling. Overexpression of Emx2 does not influence scapula blade formation but leads to the development of an additional posterior digit in the anterior border of the limb. Taken together, the data presented implicate that Emx2 expression is necessary but not sufficient for the development of the scapula blade. It is not a marker for scapula development but rather provides positional information along the proximodistal and anterior-posterior limb axes, whereas the specificity of the developing skeletal elements is determined by the concerted interaction of Emx2 with other factors.

Control of the temporal and spatial Uncx4.1 expression in the paraxial mesoderm of avian embryos.

Uncx4.1 is a homeobox containing transcription factor that determines the development of the pedicles of the neural arches, transverse processes and proximal ribs. In this paper we characterize the expression pattern of Uncx4.1 during chick embryogenesis with special focus on its expression in the paraxial mesoderm. In the presomitic mesoderm, Uncx4.1 is expressed in the caudal halves of the somites I and II. In the newly formed somites, Uncx4.1 expression remains in the caudal somite halves but becomes restricted to the somitocoele and the ventral epithelial wall. After somite compartmentalization, Uncx4.1 is expressed in the caudal half of the sclerotome in a well defined spatial and temporal pattern. Micromanipulations revealed that Uncx4.1 expression in the presomitic mesoderm is independent of signals from the axial structures and presumably induced by the intrinsic Notch/Delta driven oscillator activity that determines craniocaudal somite polarity. In contrast, in the maturing somite Uncx4.1 expression depends on signals from the axial structures. The notochord-floor plate complex is essential for maintaining Uncx4.1 expression in the caudal somite half. The neural tube is necessary for providing sufficient Uncx4.1 positive sclerotomal material to enable development of pedicles of the neural arches and transverse processes.

Expression of kinesin kif5c during chick development.

Kinesins are molecular motors associated with microtubules. They act mainly as intracellular transport proteins carrying different cargos like organelles along the microtubules. We cloned the avian homologue of the mammalian kif5c gene, a member of the khc family coding for the heavy chain of conventional kinesin. Its murine homologue has been described to be specific for neuronal tissue. Here we present the expression pattern of kif5c in chick embryos. We found a highly dynamic expression pattern for kif5c in a variety of developing tissues including neuronal and mesodermal tissues. In young embryos the expression pattern around Hensen's node is asymmetric with stronger expression on the right side, implying that kif5c is involved in the formation of the left-right body axis. A connection with intracellular transport linked to early asymmetric morphogenesis in the node is likely. Vesicles containing signaling molecules could be possible cargos. At later stages, kif5c expression is found in the paraxial, intermediate and somatic mesoderm and in the tail bud. The expression in the paraxial mesoderm occurs first during segmentation and continues in the epithelial somites and the dermomyotome. During neurulation kif5c is expressed in ectodermal and neural-plate cells. In older embryos, the expression is restricted to the dorsal root and cranial ganglia, neural tube and olfactory tract. Taken together, our results demonstrate that in the chick embryo, kif5c plays a role during different morphogenetic processes.

Cloning and characterization of a trypsin-encoding cDNA of the human body louse Pediculus humanus.

From a cDNA library of the whole insect, a trypsin gene of Pediculus humanus has been cloned and sequenced. The 908 bp clone has an open reading frame of 759 bp, which encodes a pre-proenzyme with 253 amino acid residues. A sixteen-residue N-terminal signal peptide is followed by a twelve-residue activation peptide with putative cleavage sites at Gly16 and Tyr28. The deduced amino acid sequence has several features typical of trypsin proteases and an overall identity of 35-43% with the trypsins of several haematophagous Diptera. The 1.0 kb genomic trypsin gene contains three introns of 102, 79 and 80 nucleotides following the codons for Gly16, Gln74 and Ala155, respectively. Only a single gene seems to be present. In Northern blot analysis, unfed first instar larvae have an identical or slightly lower level of trypsin mRNA than fed adult lice, and in adults 2-24 h after the bloodmeal this gene shows a constitutive expression. After in vitro transcription and translation, the activation peptide is cleaved by chymotrypsin, a so far unreported phenomenon in trypsin activation.

Glycosaminoglycans and proteoglycans in the skin of aneuploid fetuses with increased nuchal translucency.

BACKGROUND: First trimester increased fetal nuchal translucency is associated with fetal aneuploidies. One of the mechanisms of pathophysiology could be an abnormal extracellular matrix facilitating the formation of an interstitial edema. A previous study investigating interstitial edema in first trimester fetuses found large amounts of hyaluronan in the skin of fetuses with trisomy 21. The aim of this study was to establish distribution patterns for a number of other glycosaminoglycans-dermatan, heparan and keratan sulphate, chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan-in the nuchal skin of normal and chromosomally abnormal fetuses at 11-14 weeks. We also investigated whether biglycan (BGN), which is located on chromosome X, is underexpressed in fetuses with Turner syndrome. Decorin (DCN), a similar-sized proteoglycan located on chromosome 12, was taken as a control. METHODS: We studied the distribution and concentration of various extacellular matrix components using immunohistochemistry, a double staining technique, in-situ hybridization, Northern and Western blot analysis. RESULTS: Chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan were increased in Turner syndrome fetuses and BGN seemed to be underexpressed compared with normal controls, while DCN was not. Dermatan, heparan and keratan sulphate showed no significant abnormal distribution in trisomies 21, 18, 13, or in Turner syndrome, compared with normal. Western and immunohistochemical analysis revealed that absence of a second X chromosome, as is the case in Turner syndrome, affects BGN protein pattern. CONCLUSIONS: An abnormal amount of glycosaminoglycans and proteoglycans presumably contributes to increased nuchal translucency.

Temporal and spatial protease nexin 1 expression during chick development.

Protease nexin 1 (Pn-1) or glia derived nexin is a secreted protease inhibitor. By screening a chick embryonic cDNA library, we isolated Pn-1 cDNA and analyzed its expression pattern during development by in situ hybridization. Pn-1 was first observed at HH-stage 3 in the primitive pit. At HH-stage 7, expression was observed in the medial part of the neural folds and asymmetrically in the right lateral plate mesoderm and at the left side of Hensen's node. At HH-stage 10-11, Pn-1 was expressed in the closing neural tube, lateral plate mesoderm and paraxial head mesoderm. From HH-stage 12 onwards, expression was observed caudally in the lateral plate mesoderm and cranially in the Wolffian duct. At the level of the compartmentalized somite, expression was seen in the sclerotome. Pn-1 was also expressed in the anterior wall of the pharynx and still in the paraxial head mesoderm. At HH-stage 15, the expression in the Wolffian duct remained caudally while the expression in the sclerotome extended along the whole body axis. A stronger expression was observed in the cranial four somites. From HH-stage 17-18 onwards, expression became visible in the mesenchyme of the developing limb buds. At these stages, expression was no longer observed in the Wolffian duct. At HH-stage 36, Pn-1 was expressed in the vertebral bodies, in the neural tube, and in the metanephros.

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.

Development of the avian lymphatic system.

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.

Upregulation of the cochaperone Mdg1 in endothelial cells is induced by stress and during in vitro angiogenesis.

Angiogenesis research has focused on receptors and ligands mediating endothelial cell proliferation and migration. Little is known about the molecular mechanisms that are involved in converting endothelial cells from a proliferative to a differentiated state. Microvascular differentiation gene 1 (Mdg1) has been isolated from differentiating microvascular endothelial cells that had been cultured in collagen type I gels (3D culture). In adult human tissue Mdg1 is expressed in endothelial and epithelial cells. Sequence analysis of the full-length cDNA revealed that the N-terminal region of the putative Mdg1-protein exhibits a high sequence similarity to the J-domain of Hsp40 chaperones. We show that this region functions as a bona fide J-domain as it can replace the J-domain of Escherichia coli DnaJ-protein. Mdg1 is also upregulated in primary endothelial and mesangial cells when subjected to various stress stimuli. GFP-Mdg1 fusion constructs showed the Mdg1-protein to be localized within the cytoplasm under control conditions. Stress induces the translocation of Mdg1 into the nucleus, where it accumulates in nucleoli. Costaining with Hdj1, Hdj2, Hsp70, and Hsc70 revealed that Mdg1 colocalizes with Hsp70 and Hdj1 in control and stressed HeLa cells. These data suggest that Mdg1 is involved in the control of cell cycle arrest taking place during terminal cell differentiation and under stress conditions.

Glomerular osteopontin expression and macrophage infiltration in glomerulosclerosis of DOCA-salt rats.

Expression of the chemoattractant osteopontin (OPN) may contribute to macrophage infiltration in many types of tubulointerstitial kidney disease, but the role of OPN in chronic glomerulosclerosis is unknown. We hypothesized that glomerular OPN expression and macrophage infiltration occur in deoxycorticosterone acetate (DOCA)-salt glomerulosclerosis in rats. Uninephrectomized rats receiving DOCA pellets and 1% saline were compared with control rats. OPN mRNA was determined by Northern blot, and OPN protein was determined by Western blot. The localization of OPN was studied by in situ hybridization and double immunohistochemistry with glomerular cell markers. Macrophage infiltration was quantified by counting ED-1-positive cells, and semiquantitative glomerulosclerosis scores were obtained. In DOCA-salt rats, OPN mRNA in the kidney was increased 2-fold over control after 9 days and 3 weeks and 20-fold after 6 weeks. Tubulointerstitial OPN staining was apparent after 21 days of DOCA treatment. Glomerular OPN mRNA and protein was detected after 42 days in parietal and visceral epithelial cells, activated myofibroblasts, and occasionally mesangial cells. Progressive glomerular macrophage infiltration occurred during the development of DOCA hypertension, paralleling the degree of glomerulosclerosis. Glomeruli staining positive for osteopontin contained more macrophages (18.4 +/- 3.4 per cross-section) than osteopontin-negative glomeruli (3.6 +/- 0.5; P < 0.05). Glomerular OPN expression occurs in chronic hypertensive glomerulosclerosis and is associated with macrophage infiltration. The data suggest a role for OPN as a chemoattractant in hypertensive glomerulosclerosis.

Cox-2 and osteopontin in cocultured platelets and mesangial cells: role of glucocorticoids.

BACKGROUND: Glomerular inflammation is characterized by a consecutive infiltration of immunoreactive cells. To mimic the early phase of glomerular injury, a coculture system of platelets and rat renal mesangial cells was established. As prototypes, the inflammation-related proteins cyclooxygenase-2 (Cox-2) and the chemotactic protein osteopontin (OPN) were investigated. METHODS: The expression of OPN and Cox-2 mRNA and protein was determined by Northern and Western blot analyses. RESULTS: Coincubation of platelets and mesangial cells led to a rapid, transient induction of Cox-2 mRNA, which peaked at two hours, whereas OPN and monocyte chemoattractant protein-1 (MCP-1) were induced at later time points. The induction of Cox-2 mRNA was concentration dependent and highly reproducible when platelets of different donors were investigated. Partial Cox-2 induction was observed when supernatants of preactivated platelets were incubated with mesangial cells. The inhibition of the signaling pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) or interference with Gi-protein signaling partially inhibited platelet-induced Cox-2 expression. Down-regulation of protein kinase C (PKC), which is a common signaling module in many pathways leading to Cox-2 induction, almost completely abrogated platelet-induced Cox-2 expression. The time pattern of Cox-2 and OPN expression suggested that Cox-2 might play a role in OPN induction. The up-regulation of OPN was dependent on de novo protein synthesis and was induced by high levels of exogenous prostaglandin E2 (PGE2; 10 micromol/L). Endogenous PGE2, however, proved not to be essential for OPN mRNA expression, because inhibition of Cox activity did not change OPN mRNA levels. Dexamethasone inhibited Cox-2 mRNA induction but increased OPN mRNA and protein expression. CONCLUSION: These data indicate that Cox-2 and OPN are independently up-regulated upon interaction of platelets and mesangial cells.

Induction of the blood-brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos.

Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood-brain barrier (BBB)-forming endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of chick embryos. We inoculated each 3-5 x 10(6) rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7 (but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin, studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy, vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated. Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential common functions of neurothelin in endothelial cells of the CNS and tumors are discussed.

Alpha8 integrin in glomerular mesangial cells and in experimental glomerulonephritis.

BACKGROUND: Mesangial cell (MC) proliferation and extracellular matrix accumulation are typical responses of renal glomeruli to injury. Extracellular matrix components are known to affect MC behavior, which is mediated primarily via integrin receptors of the beta1 family. In addition to alpha1, alpha3, alpha5, and alpha6 chains of beta1 integrins, recent studies have shown the alpha8 chain to be expressed in glomeruli and renal vasculature. alpha8beta1 can serve as a receptor for fibronectin, which is abundant in the mesangium. We investigated the glomerular expression pattern of the alpha8 chain in renal tissues of mouse, rat, and humans as well as in cultured MCs. In addition, the regulation of alpha8 expression in MCs was studied in culture and in nephritic rats. METHODS: The expression of alpha8 protein in kidney tissue and cultured MCs was investigated by immunohistochemistry, immunocytochemistry, and Western blotting. The effects of TGF-beta1 on alpha8 mRNA levels in MCs were studied by Northern blot analysis. In addition, time course studies of glomerular abundance and localization of alpha8 were performed in rats with mesangioproliferative anti-Thy1.1 nephritis. RESULTS: In tissue sections of normal human, rat, and mouse kidney, we found strong immunohistochemical staining for alpha8 in the mesangium and in the media of renal arterioles. Double staining for alpha8 and Thy1.1, a surface antigen of rat MCs, showed alpha8 to be specifically expressed in MCs but not in glomerular endothelial and epithelial cells. In anti-Thy1.1 nephritis of rats, the glomerular abundance of alpha8 protein was reduced in the early mesangiolytic phase but was increased greatly with subsequent MC proliferation, peaking at day 6 of disease. At later stages of this reversible form of nephritis, the number of MCs and the extent mesangial alpha8 staining declined to control levels. Cell culture experiments revealed that freshly plated MCs organize alpha8 into focal contacts within one hour after attachment to fibronectin and vitronectin substrata, showing colocalization with focal contact proteins vinculin and talin. Stimulation of MCs with transforming growth factor-beta1 led to increases of alpha8 mRNA and protein levels. CONCLUSIONS: These results show that in human, rat, and mouse glomeruli, alpha8 integrin is strongly and exclusively expressed in MCs. Gene expression of alpha8 is regulated in cultured MCs, and alpha8 protein abundance is regulated in vivo and in MC culture. It is currently unclear what functional properties this integrin receptor protein has with regard to MC anchorage to extracellular matrix and modulation of the MC phenotype in normal and diseased glomeruli. However, in view of its abundance in the mesangium, alpha8beta1 integrin could be an important MC receptor of matrix ligands and may play a role in the embryology, physiology, and pathophysiology of the glomerular capillary tuft.

Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo.

BACKGROUND: Thrombospondin 1 (TSP1), a multifunctional, matricellular glycoprotein, is expressed de novo in many inflammatory disease processes, including glomerular disease. Short peptide fragments derived from the type I properdin repeats of the TSP1 molecule mimic anti-angiogenic and/or transforming growth factor-beta (TGF-beta)-activating properties of the whole TSP1 glycoprotein. We investigated the effects of D-reverse peptides derived from the type I domain of TSP1 in experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), as well as their effects on cultured mesangial and glomerular endothelial cells. METHODS: Effects of TSP peptides on proliferation of mesangial or glomerular endothelial cells in culture after growth arrest or growth factor stimulation (fibroblast growth factor-2, platelet-derived growth factor-BB, 10% fetal calf serum) were measured by [3H]thymidine incorporation assay. Adhesion of rat mesangial cells (MCs) to a TSP-peptide matrix was assayed using an attachment-hexosaminidase assay. TSP peptides were intraperitoneally injected daily in rats that had received an intravenous injection of polyclonal anti-Thy1 antibody to induce mesangial proliferative glomerulonephritis. On biopsies from days 2, 5, and 8 of anti-Thy1 disease, mesangial and glomerular endothelial proliferation, matrix expansion, mesangial activation, and microaneurysm formation were assessed. Functional parameters such as blood pressure and proteinuria were also measured. RESULTS: An 18-amino acid peptide (type I peptide) with anti-angiogenic and TGF-beta-activating sequences decreased mesangial and glomerular endothelial cell proliferation in vitro and in vivo and reduced microaneurysm formation and proteinuria in experimental glomerulonephritis. Analogues lacking the TGF-beta-activating sequence mimicked most effects of the type I peptide. The mechanism of action of these peptides may include antagonism of fibroblast growth factor-2 and alteration of MC adhesion. The TGF-beta-activating sequence alone did not have significant effects on mesangial or glomerular endothelial cells in vitro or in experimental kidney disease in vivo. CONCLUSION: Peptides from TSP1 may be promising therapeutics in treating glomerular disease with mesangial and endothelial cell injury.

Mesangial cells and their adhesive properties.

Glomerular mesangial cells play a central role in maintaining structure and function of the glomerular capillary ultrafiltration apparatus. Under physiological and pathological conditions, mesangial cells regulate amount and composition of the surrounding extracellular matrix. Conversely, components of the embedding matrix affect the mesangial cell phenotype. These interactions are mediated via specific cell surface receptors, the best studied group of which is the beta1 integrin family. The beta1 integrins play a role in mesangial cell adhesion, migration, survival and proliferation. Expression and abundance of integrins in healthy and diseased glomeruli and their functions and mediation of signals are discussed in this review. Other factors modulating mesangial cell-matrix interactions, such as antiadhesive proteins, cytokines, disintegrins and nitric oxide, are also considered. The available evidence from in vitro and in vivo studies indicates that receptor-mediated interactions between mesangial cells and the normal or abnormal extracellular matrix regulate the mesangial cell phenotype and thus contribute to normal maintenance of the glomerulus and to remodeling and repair of the glomerular capillary tuft in response to injury.

Differential expression of osteopontin, PC4, and CEC5, a novel mRNA species, during in vitro angiogenesis.

The formation of new capillaries from preexisting blood vessels, a process termed angiogenesis, plays a key role in many physiological and pathological conditions such as wound healing, embryogenesis, and tumor growth. The aim of this study was to identify changes in endothelial cell gene expression specifically associated with angiogenesis. Using an in vitro model and the differential display strategy, we compared gene expression patterns of rat microvascular endothelial cells cultured in two (2D) and three-dimensional (3D) culture. In 2D culture, the cells express actin and proliferate, whereas in 3D culture actin expression is downregulated, and the cells are mitotically quiescent and reorganize into vascular tubes. We identified three differentially expressed genes, osteopontin, PC4, and CEC5, a novel mRNA species, with homology to calmodulin-dependent protein kinases. The expression patterns were confirmed by Northern blot analysis. In conclusion, the analysis of gene expression in endothelial cells in 3D and 3D culture allows the identification of genes differentially expressed during angiogenesis. These genes or proteins may serve as targets for therapeutic modulation of angiogenesis.

Exogenous nitric oxide inhibits mesangial cell adhesion to extracellular matrix components.

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.

Regulation of osteopontin expression in rat mesangial cells.

Hypercellularity and accumulation of extracellular matrix are common responses of renal glomeruli to inflammatory stimuli. Using the differential display approach, we compared the gene expression patterns of proliferating and differentiating rat mesangial cells in two- and three-dimensional cultures. Osteopontin, an extracellular matrix protein, was found to be transcribed, synthesized, and secreted by rat mesangial cells. Osteopontin transcription was not associated with cell proliferation and was found to be FCS-inducible in proliferating cells. Osteopontin expression was independent of exogenously supplied FCS in differentiating cells. The presented data indicate that osteopontin is differentially regulated in proliferating and differentiating mesangial cells.