(73/1076) Injury induces dedifferentiation of smooth muscle cells and increased matrix-degrading metalloproteinase activity in human saphenous vein.
Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure. (+info)
(74/1076) Pathological analysis of local delivery of paclitaxel via a polymer-coated stent.
BACKGROUND: Paclitaxel can inhibit vascular smooth muscle proliferation in vitro, and early studies suggest that paclitaxel may be useful in preventing restenosis. Early and late intimal growth and local vascular pathological changes associated with paclitaxel delivered via stents have not been fully explored. METHODS AND RESULTS: Localized drug delivery was accomplished with balloon-expandable stainless steel stents coated with a cross-linked biodegradable polymer, chondroitin sulfate and gelatin (CSG), containing various doses of paclitaxel. CSG-coated stents with paclitaxel (42.0, 20.2, 8.6, or 1.5 microgram of paclitaxel per stent), CSG-coated stents without paclitaxel, and uncoated stents (without paclitaxel or CSG) were deployed in the iliac arteries of New Zealand White rabbits, which were killed 28 days after implant. Mean neointimal thickness at stent strut sites was reduced 49% (P<0.0003) and 36% (P<0.007) with stents containing 42.0 and 20.2 microgram of paclitaxel per stent, respectively, versus CSG-coated stents without paclitaxel. However, histological findings suggested incomplete healing in the higher-dose (42.0 and 20.2 microgram) paclitaxel-containing stents consisting of persistent intimal fibrin deposition, intraintimal hemorrhage, and increased intimal and adventitial inflammation. Stents coated with CSG alone (without paclitaxel) had similar neointimal growth as uncoated stents. In a separate group of rabbits killed at 90 days, neointimal growth was no longer suppressed by CSG-coated stents containing 42.0 or 21.0 microgram of paclitaxel CONCLUSIONS: CSG coating appears to be a promising medium for localized drug delivery. Paclitaxel polymer-coated stents reduce neointima formation but are associated with evidence of incomplete healing at 28 days. However, neointimal suppression was not maintained at 90 days. (+info)
(75/1076) Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris.
A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris. Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the fact that gelatins are highly susceptible to proteolysis because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry. Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed. Esterification of the carboxylic amino acid side chains resulted in normal migration. The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C. The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins. (+info)
(76/1076) Bronchial stump reinforcement in right pneumonectomy with fascia lata and gelatin resorcin formalin (GRF) glue: case report.
We reinforced the bronchial stump with fascia lata and Gelatin Resorcin Formalin (GRF) glue in a right pneumonectomy. This method was found to be simple and useful. We describe our case and the method herein. A 62-year-old woman had a malignant polypoid lesion which completely occluded the introitus of the right main bronchus and deviated to the introitus of the left main bronchus. Right pneumonectomy was done but materials (pleura, pericardium, intercostal muscle, etc.) obtained from the thoracic cavity were insufficient for bronchial stump reinforcement due to severe adhesion caused by prior tuberculosis. Therefore, we reinforced the bronchial stump using the fascia lata and GRF glue. Fascia lata is a superior material for reinforcement in terms of strength and ease of molding, as well as harvesting. GRF glue is a superior adhesive with rapid and strong fixation. We consider this method of reinforcing the bronchial stump with fascia lata and GRF glue to be feasible, in particular, for pneumonectomy or lobectomy without adequate material in the thoracic cavity because of severe adhesion or lesions. (+info)
(77/1076) Development and application of a quantitative, specific assay for Cryptosporidium parvum oocyst detection in high-turbidity environmental water samples.
Chlorine-resistant Cryptosporidium parvum oocysts in drinking water play an important role in the epidemiology of cryptosporidiosis. Current methods of detecting these organisms in water are insensitive, labor-intensive, highly subjective, and severely limited by sample turbidity. We describe here an alternative technique utilizing electrochemiluminescence (ECL) technology for detecting C. parvum oocysts in environmental water samples. This method is quantitative, reproducible, and requires only minimal sample processing. Currently, the ECL assay can detect as few as one oocyst in one milliliter of concentrated test sample with sample turbidity of up to 10,000 nephelometric turbidity units. Water and sewer samples collected during a cryptosporidiosis outbreak were tested by ECL assay. Cryptosporidium parvum oocysts were found in the source water at the time of outbreak, and a sharply decreasing level of oocysts in sewer samples was observed over a three-month period following the outbreak. (+info)
(78/1076) Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats aged 0-21 days: II. Two proteinases, gelatinase B and collagenase-3, are implicated in the lysis of collagen fibrils.
In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-microm-thick band of matrix, referred to as "pre-resorptive layer." This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti-gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10-21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer-most likely through a cooperative attack leading to the disintegration of the collagen fibril framework. (+info)
(79/1076) Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin.
Expression of tissue transglutaminase (transglutaminase II, tTG) was shown to increase drastically during monocyte differentiation into macrophages; however, its role in monocytic cells remains largely unknown. This study describes a novel function of cell surface tTG as an adhesion and migration receptor for fibronectin (Fn). Two structurally related transglutaminases, tTG and the A subunit of factor XIII (FXIIIA), are expressed on the surface of monocytic cells, whereas only surface tTG is associated with multiple integrins of the beta1 and beta3 subfamilies. Both surface levels of tTG and the amounts of integrin-bound tTG are sharply up-regulated during the conversion of monocytes into macrophages. In contrast, a reduction in biosynthesis and surface expression of FXIIIA accompanies monocyte differentiation. Cell surface tTG is colocalized with beta1- and beta3-integrins in podosomelike adhesive structures of macrophages adherent on Fn. Down-regulation of surface tTG by expression of antisense tTG construct or its inhibition by function-blocking antibodies significantly decreases adhesion and spreading of monocytic cells on Fn and, in particular, on the gelatin-binding fragment of Fn consisting of modules I6II1,2I7-9. Likewise, interfering with the adhesive function of surface tTG markedly reduces migration of myeloid cells on Fn and its gelatin-binding fragment. These data demonstrate that cell surface tTG serves as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing Fn matrices during inflammation. (+info)
(80/1076) Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion.
Activation of proMMP-2 by MT1-MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP-2 bound to MT1-MMP on the cell surface acts as a receptor for proMMP-2. Subsequently, adjacent TIMP-2-free MT1-MMP activates the proMMP-2 in the ternary complex. In this study, we demonstrate that MT1-MMP forms a homophilic complex through the hemopexin-like (PEX) domain that acts as a mechanism to keep MT1-MMP molecules close together to facilitate proMMP-2 activation. Deletion of the PEX domain in MT1-MMP, or swapping the domain with the one derived from MT4-MMP, abolished the ability to activate proMMP-2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1-MMP lacking the catalytic domain (MT1PEX-F) efficiently inhibited complex formation of the full-length enzymes and activation of pro MMP-2. Furthermore, expression of MT1PEX-F inhibited proMMP-2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1-MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue. (+info)