Current results of total endovascular repair of thoracoabdominal aortic aneurysms.

Minimally invasive surgical solutions for patients with extensive aortic disease are eagerly awaited, since open repair is often associated with high rates of morbidity and mortality. In the last decade, the development of fenestrated and branched aortic endografts has offered a therapeutic option to patients deemed unsuitable for major surgery. Preliminary studies showed promising early results, while mid- and long- term data are scarce. The aim of this paper was to review current results of total endovascular repair of thoracoabdominal aortic aneurysms (TAAA) with a single model of endograft in the published literature. A literature search was conducted, and our two-center experience with fenestrated and branched endografts in the treatment of TAAA, with the Cook Zenith endograft, is presented. Early results show perioperative mortality rates ranging from 0% to 21%, spinal cord ischemia from 0% to 33.3%. At a mean follow up ranging from 9 to 19 months, reinterventions are needed in 3.3% to 25% of the cases, with a mid term visceral branch patency of 90% to 100%. Current experiences with total endovascular TAAA repair show promising results, in selected centers with large experience in complex aortic endografting. With increasing follow- up times, need for reintervention is growing, while aneurysm-related deaths remain rare. Long-term results are still lacking, but these encouraging data and further technological developments will support wider adoption of the technique.

Role of the basement membrane in tumor cell dormancy and cytotoxic resistance.

OBJECTIVES AND METHODS: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). RESULTS: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. CONCLUSIONS: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

[Coarctation of he abdominal aorta]. / Coarctation of the abdominal aorta.

The Authors report a 49-year-old woman complaining of slight nocturnal lower limb pain in whom an uncommon type IV coarctation of the infrarenal aorta associated with multiple renal arteries, slight hypoplasia of iliac and femoral arteries bilaterally, and a retroaortic left vein were found. She underwent an aorto-aortic prosthetic repair. The correction of this vascular condition was followed by partial improvement of her symptoms. The suspicion of an associated ischaemic spinal origin of these painful symptoms may be suggested by the typical and often complex presence of multiple vascular malformations described in patients with coarctation of the abdominal aorta.

Inhibition of DNA topoisomerase I activity by heparan sulfate and modulation by basic fibroblast growth factor.

Eukaryotic DNA topoisomerase I catalyzes changes in the superhelical state of duplex DNA by transiently breaking single strands thereby allowing relaxation of both positively and negatively supercoiled DNA. Topoisomerase I is a nuclear enzyme localized at active sites of transcription, and abnormal levels of the enzyme have been observed in a variety of neoplasms. Because the enzyme binds heparin and, given the presence of heparan sulfate within the nuclei of mammalian cells, we sought to investigate the interaction between topoisomerase I and sulfated glycosaminoglycans isolated from normal and neoplastic human liver. The results demonstrated that low concentrations (approximately 100 nM) of heparan sulfate from normal liver but not from its malignant counterpart effectively blocked relaxation of supercoiled DNA driven by either purified holoenzyme or topoisomerase I activity present in nuclear extracts of three malignant cell lines. Heparin acted at even lower (approximately 10 nM) concentrations. Moreover, we show that basic fibroblast growth factor could interfere with this heparan sulfate/heparin-driven inhibition and that both basic fibroblast growth factor and heparin-binding sites co-localized in the nuclei of U937 leukemic cells. Our results suggest that DNA topoisomerase I activity may be modulated in vivo by specific heparan sulfate moieties present in normal cells but markedly reduced or absent in their transformed counterparts.

The antiproliferative action of a melphalan hexapeptide with collagenase-cleavable site.

PURPOSE: The objective of the present study was to examine the relevance of collagenase in the antitumor action of a melphalan peptide (MHP) with a collagenase-cleavable sequence. The question was addressed as to whether collagenase may act as an activator or a target in the antiproliferative mechanism of MHP. METHODS: Melphalan was inserted into peptides representing the sequence Pro-Gln-Gly-Ile-Ala.Gly of the collagenase-cleavable site in collagens. Changes in growth and collagenase IV activities of HT-1080, HT-29, HT-168, and MCF-7 cell cultures were investigated. RESULTS: The present investigations provide data indicating that Pro-Gln-Gly-Ile-Mel-Gly (melphalan hexapeptide, MHP) is a substrate for both bacterial and 72-kDa type IV collagenases and that in this way it can generate Ile-Mel-Gly (melphalan tripeptide, MTP) of higher cytotoxic potency. Indeed, the formation of MTP was detected in the conditioned medium of HT-1080, a collagenase IV-producing human fibrosarcoma. In a comparison of equimolar concentrations of melphalan and its two peptide derivatives (MHP and MTP), superior antiproliferative action of MTP was seen in HT-29, HT-1080, and HT-168 tumor cell cultures. However, the relatively modest cytostatic actions of MHP were increased when bacterial collagenase was added to the cell cultures. After melphalan treatment, reduced levels of both 92 and 72-kDa type IV collagenases were seen in the HT-1080 cell cultures. However, the reduction of collagenase activity and the cell counts did not run parallel in the MTP- or MHP-treated cultures; indeed, collagenase activity related to cell numbers showed an elevated level. CONCLUSIONS: As the conversion of MHP to the more toxic MTP was detected in the presence of collagenases, it is possible that collagenase-directed activation of prodrugs may be a promising approach for the development of more selective cytostatic drugs against malignant tumors with high collagenase activities.

Role of sinusoidal heparan sulfate proteoglycan in liver metastasis formation.

Previous studies have indicated that the predominant sites of tumor cell extravasation in the liver are the sinusoidal vessels, where tumor cells contact the sinusoidal endothelium and the subendothelial extracellular matrix containing the basic components of the basement membrane. We studied the role of sinusoidal extracellular matrix in metastatsis formation by 3LL-HH murine tumor cells selected for their preferential liver colonization. 3LL-HH tumor cells did not efficiently adhere to cryosections of the liver, but they recognized the sinusoids and vessel walls. Pre-treatment of the mice with polyclonal anti-basement membrane antibodies [anti-laminin, anti-fibronectin and anti-heparan sulfate proteoglycan (HSPG)] significantly modulated the organ distribution of tumor cell colonies following intracardial injection: all 3 antibodies inhibited kidney colonization; anti-laminin and anti-fibronectin antibodies inhibited lung colonization; and only anti-HSPG antibody inhibited liver colonization. In several organs such as the heart, stomach, pancreas and bladder, anti-basement membrane antibody treatment did not alter the process of colonization. Immunofluorescence studies showed that anti-HSPG antibody recognized the basement membranes of sinusoids and blood vessels. Our data suggest a specific involvement of sinusoidal HSPG in the liver colonization of 3LL-HH cells.

Characterization of a basement membrane protein (BM180) using capillary electrophoresis.

Coeliac disease (gluten intolerance) is a genetically based autoimmune disease that becomes evident following ingestion of cereal prolamins such as the wheat gliadins. The process of this disease is not yet thoroughly understood. Purification of basement membrane protein-180 (BM180) (a basement membrane protein with a potential autoantigen role) and multiple analysis of the purified protein can lead to a better understanding of this disorder, which affects millions of people worldwide. Our preliminary work on the purification and characterization of this protein is presented in this paper. BM180 proteins were expressed in mouse EHS (Engelberth-Holm-Swarm) tumor cells. A crude purification process (see "Methods") was performed and the purified fractions were analyzed by capillary gel electrophoresis (CGE) for determination of the apparent molecular weight of the protein components in each fraction. The fractions, which contained compounds of the expected molecular weight, were further analyzed using a capillary zone electrophoresis (CZE) method developed for the routine analysis of wheat gliadins. Using the two CE methods, we were able to compare BM180 with certain gliadin fractions. Additional information on the protein stability was also obtained.

The antimetabolite Tiazofurin (TR) inhibits glycoconjugate biosynthesis and invasiveness of tumour cells.

We investigated the effect of Tiazofurin (TR-2-beta-D-furanosylthiazole-4-carbamide) on tumour cell invasion using metastatic 3LL-HH murine lung carcinoma and HT168-M1 human melanoma as experimental models. TR pretreatment of 3LL-HH cells, in a dose range of 15-60 microM, caused inhibition of cell proliferation, adhesion to plastic and extracellular matrix proteins. The TR-induced altered matrix interactions of 3LL-HH cells were reflected in decreased migration through matrix-covered filters. Analysis of the expression of certain invasion markers indicated that TR suppressed the expression of alpha v beta 3 integrin and MMP2 metalloproteinase. Biochemical studies indicated that 24 h 60 microM TR treatment of 3LL-HH cells inhibited glycosylation of a wide range of glycoproteins with the most pronounced effect on proteoglycans. TR pretreatment of 3LL-HH tumour cells resulted in the loss of lung colonisation potential in vivo. Furthermore, in vivo TR treatment inhibited the formation of liver metastases of 3LL-HH murine carcinoma. TR treatment also induced inhibition of integrin and MMP2 expression, migration and liver colonisation of the human melanoma HT168-M1 cell line. Since the TR concentration which inhibited various cellular functions was much lower for cell adhesion and lung colonisation than for cell proliferation, we suggest that the predominant effect of TR is the inhibition of metastasis in these model systems. We also suggest that both the effect of TR on tumour cell proliferation and on extracellular matrix interaction contribute to its remarkable antimetastatic potential in vivo.

Immunological and partial sequence identity of mouse BM180 with wheat alpha-gliadin.

BM180, a novel 180-kDa basement membrane protein enriched in guanidine-HCl extracts of lacrimal and parotid exocrine secretory glands, was immunopurified using the secretion inhibitory monoclonal antibody 3E12. The N-terminal amino acid sequence was found to be VRVPVPQLQPQNP. An identical sequence comprises the N-terminus of the wheat storage protein alpha-gliadin. The presence of a gliadin-like protein in basement membranes was confirmed using a monoclonal and several polyclonal anti-gliadin antibodies, the former of which detected a 180-kDa protein in basement membrane blots. A full-length alpha-gliadin cDNA was found to hybridize at high stringency with mouse and human genomic DNA; and in lacrimal gland Northern blots with a 2.3-kb message. Since BM180 appears to be required for stimulus-secretion coupling by lacrimal acinar cells, circulating anti-alpha-gliadin antibodies associated with Sjögren's syndrome ('Dry Eye') and more commonly in Coeliac disease, may be secretion inhibitory.

Modulation of heparan-sulphate/chondroitin-sulphate ratio by glycosaminoglycan biosynthesis inhibitors affects liver metastatic potential of tumor cells.

Previous data have indicated that the proteoglycan (PG) pattern is different on tumor cells with different liver metastatic potential. We selected "conventional" glycosaminoglycan (GAG) biosynthesis inhibitors, beta-D-xyloside (BX), 2-deoxy-D-glucose (2-DG), ethane-l-hydroxy-l,l-diphosphonate (ETDP) and the newly discovered 5-hexyl-2-deoxyuridine (HUdR), to modulate PGs on highly metastatic/liver-specific 3LL-HH murine carcinoma and HT168 human melanoma cells and to influence their liver colonization potential. These compounds all induced remarkable changes in GAG biosynthesis, but to varying degrees: glucosamine labelling was affected mainly by 2-DG, and HUdR and sulphation by BX and HUdR. Furthermore, the ratio of heparan sulphate/chondroitin sulphate (HS/CS) of PGs was increased by ETDP and decreased after treatment by HUdR. In addition to changes in PG metabolism, tumor-cell proliferation and adhesion to fibronectin were affected; BX and 2-DG stimulated cell proliferation and adhesion, while HUdR inhibited both proliferation and adhesion. Most interestingly, HUdR, the most effective inhibitor of HS/HSPG, depressed the formation of liver colonies, while ETDP, the most effective inhibitor of CS/CSPG, stimulated the appearance of liver colonies. These observations indicated that, at least in these experimental systems, tumor cells with a high HS/CS ratio are more likely to form liver metastases; consequently, anti-HS agents could also be anti-metastatic.

The in vitro interaction of proteoglycans with type I collagen is modulated by phosphate.

Binding of proteoglycans to type I collagen in vitro was assessed using radiolabeled decorin, biglycan, and large proteoglycans and acid-extracted bovine tendon collagen. Decorin, biglycan, and large proteoglycans were all bound to collagen fibrils in phosphate-buffered saline (PBS) containing 3 mM sodium phosphate. Only decorin was bound when the phosphate concentration in PBS was increased to 30 mM. These distinct binding characteristics were not altered by the presence of 10% serum, by purification of the proteoglycans in 7 M urea and 4 M guanidine HCl, or by digestion of the collagen with pepsin. In addition to being affected by phosphate, both glycosaminoglycan and proteoglycan binding to collagen was inhibited by sulfate, an anion with similar structure, and by molecules that contain sulfate or sulfonate groups, such as chondroitin sulfate and N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (Tes). The rate of in vitro collagen fibrillogenesis was retarded by increasing concentrations of phosphate. Decorin decreased the rate of collagen fibrillogenesis in all buffers and virtually abolished fibril formation when added in buffer containing both 30 mM phosphate and 30 mM Tes. It is concluded that decorin binds to collagen through interaction between collagen and the decorin core protein, whereas biglycan and large proteoglycans bind to collagen fibrils through their glycosaminoglycan chains. This glycosaminoglycan-collagen interaction is inhibited by phosphate, sulfate, and sulfonate. These observations may clarify contradictory results among previous in vitro studies of proteoglycan-collagen interaction. Since the phosphate concentration of blood and interstitial fluid is estimated to be approximately 1 mM, collagen-glycosaminoglycan interactions could occur in tissue.

Aggrecan in bovine tendon.

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Atomic force microscopy of mammalian sperm chromatin.

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, ellipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.

Morphometric analysis of intact sperm heads and of sperm nuclei in the mouse.

A morphometric analysis of mouse sperm and of their nuclei was undertaken to investigate their respective post-testicular maturation. Sperm were collected from the testis, caput and cauda epididymidis, and their corresponding nuclei were isolated. Results indicate that the post-testicular maturation of sperm is distinct from that of nuclei. The size of intact sperm heads increases in the caput followed by a subsequent decrease in the cauda. In contrast, sperm nuclei decrease progressively in size. In general, a greater magnitude and number of alterations in intact heads and nuclei occur while in transit from the testis to the caput than during passage to the cauda epididymis. These results suggest that the period immediately following their release from the testis is crucial to the complete morphological maturation of sperm heads and nuclei.

Proteoglycans in the compressed region of human tibialis posterior tendon and in ligaments.

Proteoglycan content and tissue morphology were examined in tendons and ligaments from 24 cadavers, ranging in age at the time of death from 1.5 months to 83 years. The region of the human tibialis posterior tendon that passes under the medial malleolus was characterized by cells having a rounded shape, positive staining with alcian blue, and higher glycosaminoglycanuronic acid content than in the more proximal region of the same tendon. Analysis of proteoglycans by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the predominant small proteoglycan of the proximal/tensional region was decorin, whereas two types of small proteoglycans (decorin and biglycan) and large proteoglycans were present in the region passing under the medial malleolus and presumably subjected to compressive and shear forces in addition to tension. The pattern of proteoglycan accumulation in the compressed region of tendon was basically similar for all individuals and showed no distinctive trends related to age after puberty. In terms of type and amount of proteoglycan, the patellar tendon was like the tensional region of the tibialis posterior. Glycosaminoglycan content in the lateral collateral ligament and anterior cruciate ligament, however, was twofold higher than in the tendons. The ligaments contained large as well as small proteoglycans, just as in the compressed region of tendon.

The interaction of decorin core protein fragments with type I collagen.

To further define the molecular interaction between decorin and type I collagen we generated a 20 kD fragment containing the N-terminal half of the core protein by Endoproteinase Arg C digestion and a 40 kD fragment including all leucine-rich repeats in the central part of decorin core by cleavage with 2-nitro-5-thiocyanobenzoate. The fragments did not influence collagen fibril formation, even at high concentration, and radioactive fragments showed little binding to collagen fibrils. Our observations suggest that neither the N-terminal half nor the central leucine-rich repeats of the decorin core protein can, by itself, interact fully with fibrillar collagen.

Quantitative fluorometry of abnormal mouse sperm nuclei.

An extensive quantitative analysis of deformed mouse spermatozoa was undertaken. Improvements over previous studies included the isolation and purification of sperm nuclei, a multifaceted analytical approach using several fluorochromes and the analysis of individual nuclei classified into shape categories. Malformed sperm nuclei in BALB/c mice could not be distinguished from normal ones in terms of total and basic proteins, sulfhydryl and disulfide group concentration, DNA concentration and chromatin organization. The shape of sperm nuclei is therefore probably determined by the manner in which the internal biochemical components are assembled.