Collagenase Content Research Articles

Inhalable nano-structured microparticles for extracellular matrix modulation as a potential delivery system for lung cancer

The use of inhalable nanoparticulate-based systems in the treatment of lung cancer allows for efficient localized delivery to the lungs with less undesirable systemic exposure. For this to be attained, the inhaled particles should have optimum properties for deposition and at the same time avoid pulmonary clearance mechanisms. Drug delivery to solid tumors is furthermore challenging, due to dense extracellular matrix (ECM) formation, which hinders the penetration and diffusion of therapeutic agents. To this end, the aim of the current work is to develop an ECM-modulating nano-structured microparticulate carrier, that not only enables the delivery of therapeutic nanoparticles (NPs) to the lungs, but also enhances their intratumoral penetration. The system is composed of acetalated maltodextrin (AcMD) NPs embedded into a water-soluble trehalose/leucine matrix, in which collagenase was loaded with different mass concentrations (10 %, 30 % and 50 %). The collagenase-containing AcMD nano-structured microparticles (MPs) exhibited suitable median volume diameters (2.58 ± 1.35 to 3.01 ± 0.68 µm), hollow corrugated morphology, sufficient redispersibility, low residual moisture content (2.71 ± 0.17 % to 3.10 ± 0.20 %), and favorable aerodynamic properties (Mass median aerodynamic diameter (MMAD): 1.93 ± 0.06 to 2.80 ± 0.10 µm and fine particle fraction (FPF): 68.02 ± 6.86 % to 69.62 ± 2.01 %). Importantly, collagenase retained as high as 89.5 ± 6.7 % of its enzymatic activity after spray drying. MPs containing 10 % mass content of collagenase did not show signs of cytotoxicity on either human lung adenocarcinoma A549 cells or lung MRC-5 fibroblasts. The nanoparticle penetration was tested using adenocarcinoma A549/MRC-5 co-culture spheroid model, where the inclusion of collagenase resulted in deeper penetration depth of AcMD-NPs.

Assessing the effectiveness of turmeric and tamarind leaves extract cream as anti-aging in vivo

Turmeric and tamarind leaves extract creams have potential as anti-aging agents, but their use has never been studied in vivo. Therefore the purpose of this study was to determine the effectiveness of turmeric and tamarind leaves extract cream in maintaining skin quality in vivo. This research was carried out by applying turmeric and tamarind leaf extract cream to 24 volunteers (having received approval from the ethical commission). Variables observed included sebum content, pore size, pigment content, moisture or moisture content, elasticity and collagenase content. The results showed that turmeric and tamarind leaves extract cream can increase sebum content up to 31.1%, reduce pore size to 0.017 mm, increase pigment content up to 13.2%, moisture up to 54.2%, elasticity up to 21.4% and collagenase content up to 37.3%.

Suppressed Expression but Not Activity of Collagenases MMP-1 and MMP-13 in Human Renal Carcinoma

Background: Collagenases are enzymes starting collagen degradation. The role of collagenases in renal carcinoma development is not well understood. Objective: Evaluation of collagen content and collagenase expression and activity in human kidney cancers. Methods: Collagen content was measured by the hydroxyproline assay. The expression and the content of collagenases were evaluated by Western blotting and ELISA. Fluorogenic substrate was used to measure enzyme activity. Results: Collagen content significantly decreases with the progression of kidney cancer. Both collagenases are first present in high molecular complexes in both control and cancer tissue. The healthy part of the kidney contains similar amounts of both collagenases. Collagenase content decreased significantly in tumor tissue with increasing cancer stage. MMP-13 activity is much higher than that of MMP-1 in all tissues investigated. We observed increasing collagenase activity (MMP-1 and MMP-13) with increasing renal cancer grade. Conclusions: The lower content and higher activity of the collagenases investigated in cancer tissue indicate that most of these enzymes are in active form in renal carcinoma. The lower collagen content in cancer tissue can be explained at least in part by increased collagenase activity.

Molecular characterization of post-thrombotic syndrome

The post-thrombotic syndrome represents a poorly understood and significant vascular health problem. This review focuses on our current understanding of the pathogenesis of post-thrombotic syndrome. We emphasize the cellular and molecular mechanisms that are responsible for the critical components of post-thrombotic syndrome. These include the initiation of deep venous thrombosis, the pathogenesis of elevated venous pressure, and the factors responsible for nonhealing of venous stasis ulcers.

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.

Are excessive granulation tissue formation and retarded wound contraction due to decreased collagenase activity in wounds in tight-skin mice?

Wound contraction is delayed in tight-skin mice but the mechanism(s) are unknown. The purpose of this study was to investigate collagenase levels and the formation of granulation tissue in experimental wounds in tight-skin mice. One full-thickness skin excision (20 x 20 mm) was made on the back of nine tight-skin and eight normal mice. Granulation tissue analyses were performed 7 days post-operatively. The collagenase activity was determined by the use of a radiolabelled telopeptide-free collagen substrate, and the amount of granulation tissue was determined gravimetrically. Wound contraction was delayed (P < 0.001) in tight-skin mice (mean 22%) compared with normal mice (mean 46%). The collagenase activity was decreased (P < 0.05) by 40%, whereas the quantity of granulation tissue was increased (P < 0.001) by 60% in the wounds of tight-skin mice. Decreased collagenase content may provide one explanation for the delayed contraction of full-thickness wounds in tight-skin mice. Furthermore, this animal would model may prove useful in the understanding of the pathogenesis, and in exploration of treatment, of excessive granulation tissue formation during wound healing.

Changes in fluid composition on the serosal surface of jejunum and small colon subjected to venous strangulation obstruction in ponies

SUMMARY In 6 anesthetized ponies, 3 segments of jejunum and 3 segments of small colon were isolated from the peritoneal cavity in plastic bags filled with Hanks' balanced salt solution. One jejunal and 1 small colon segment were subjected to venous strangulation obstruction for 3 hours (vso-3), venous strangulation obstruction for 6 hours (vso-6), or a 6-hour sham procedure to control for changes induced by isolation in a plastic bag. Additional segments of jejunum and colon that were not placed in bags served as controls for histologic examination and collagenase measurements. Samples of fluid surrounding the intestine were obtained for chemical analyses, nucleated cell count, aerobic and anaerobic bacteriologic culture, and measurement of collagenase activity. Full-thickness tissue samples were obtained for histologic examination and measurement of collagenase content. Bacteria did not cross the intestinal wall after 3 and 6 hours of vso, despite severe mucosal lesions in these segments. At 6 hours, Po2 was significantly less and Pco2 was significantly (P&lt;0.05) greater in the fluid surrounding the vso-6 jejunal segments, compared with the sham jejunal segments. The pH was significantly (P&lt;0.05) less in fluid surrounding vso-6 small colon segments, compared with the sham colon segments at 6 hours. For jejunum and small colon, phosphate and lactate concentrations were significantly (P&lt;0.05) greater in vso-6 fluid than in the corresponding sham fluids at 6 hours. Fibrin formed around all vso segments, although fibrinogen was not detected in the surrounding fluid, indicating possible rapid conversion of fibrinogen to fibrin. Fluid collagenase activity increased significantly (P&lt;0.05) in all segments over 6 hours. The preparation used in this study was successful in measuring local changes on the serosal surface of intestine subjected to vso and in isolating segments under study in a sterile environment.

Collagenase in Wound Healing: Effect of Wound Age and Type

Collagenase is believed to be important for cell migration and collagen remodeling during tissue repair and regeneration. We have investigated collagenase concentrations in different types of surgically inflicted wounds in pigs. Collagenase was extracted from tissue homogenates of wounds by heating to 60 degrees C for 6 min in 0.1 M CaCl2. The molecular weight of latent collagenase was about 52 kDa. Activated collagenase produced the characteristic 3/4 fragment of collagen. Collagenase was assayed by the use of radiolabeled telopeptide-free collagen. To detect maximal collagenase activity, extracts were reduced and alkylated to destroy inhibitors, then activated with aminophenylmercuric acetate. Sutured incisions showed peak collagenase content on postoperative day 1 and thereafter steadily declining concentrations. Granulation tissue from non-sutured large defect full-thickness wounds showed high collagenase content on postoperative day 5 and then a sharp decline to day 7 followed by a slowly declining curve to postoperative day 21. Partial-thickness wounds exhibited a different time course, with collagenase increasing to peak concentrations on postoperative days 3-5; however, a large proportion of the detected collagenase was due to the adherent scab. By day 7 collagenase concentrations approached the low concentrations of normal skin when epithelialization was complete and the scab rejected. In general, collagenase shows an early maximum and then declines with postoperative time, with the sharpest decline occurring when epithelialization is complete.

A serum factor promotes collagenase synthesis by an osteoblastic cell line.

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.

Changes in active and latent collagenase in human placenta around the time of parturition

High levels of collagenase are present in cervical extracts and in the circulation in women at parturition. This study examines the content of collagenase in human placentas at term, the possible contribution to circulating enzyme, and the changes that occur at parturition. Active and latent forms of collagenase are detectable in placentas with apparent relative molecular mass of 60,000 and 65,000 d, respectively. Gel-filtration chromatography was used to identify the presence of excess tissue inhibitor of metalloproteinase as the major collagenase inhibitor in the extracellular matrix of human placentas. After the onset of labor, there was a significant increase in total collagenase activity. Inactivation of the tissue inhibitor of metal loproteinase by reduction of placental extracts with dithiothreitol and alkylation with iodoacetamide resulted in a twelvefold to seventeenfold increase in collagenase activity. Umbilical cord collagenase levels were significantly lower than those in maternal circulation. The possibility of circulating collagenase originating from placenta in labor is discussed.

Critical carotid stenoses: Morphologic and chemical similarity between symptomatic and asymptomatic plaques

To identify microanatomic and chemical features that may mark the transition from asymptomatic to symptomatic atherosclerotic carotid lesions, we evaluated 62 carotid artery bifurcation plaques including 45 high-grade stenoses removed at endarterectomy and 17 nonstenotic plaques recovered at autopsy. Morphologic features were evaluated on multiple-interval histologic sections and were graded for the presence of hemorrhage, ulceration, thrombosis, lumen surface irregularity, and calcification. Plaque hemorrhage, recent and remote, was found in most of the specimens, and did not discriminate between symptomatic and asymptomatic stenotic plaques. High-grade carotid stenotic plaques were associated with a significantly higher incidence of ulceration (53%), thrombosis (49%), and lumen irregularity (78%) when compared to nonstenotic asymptomatic plaques (6%, 0%, and 17%, respectively; p < 0.01). Although these features were more prominent in lesions that produced symptoms, they were present in 80% of the stenotic bifurcations, and did not distinquish between symptomatic and asymptomatic endarterectomy plaques. No significant differences were found between symptomatic and asymptomatic high-grade lesions with respect to collagen, DNA, total cholesterol, fibrinogen, lipase, elastase, or collagenase content. We conclude that intraplaque hemorrhage is commonly seen in carotid plaques even without severe stenosis, and it does not appear to be a dominant determinant of symptoms. Ulceration and surface thrombi that may lead to cerebral embolization are prominent features in markedly stenotic plaques even when symptoms are absent. The disruptive processes that underlie plaque instability appear to be closely associated with plaque size and stenosis rather than plaque composition.

The ultrastructure of osteoclasts in microphthalmic mice

Osteoclasts in microphthalmic (mi/mi) mice were significantly smaller than in their heterozygous (mi/+) or normal (+/+) littermates as measured by electronmicroscopic morphometry. Clear zones were also significantly smaller and normal ruffled borders were absent. The cytoplasm was not fully differentiated. No differences were seen between osteoclasts from mi/+ and +/+ mice. The number of osteoclasts was not different in mi/mi, mi/+ or +/+ mice. Lysosomal enzymes and collagenase content of bones in the three genotypes did not show marked differences.

Parathyroid Hormone Stimulation of Collagenase Secretion by Isolated Bone Cells*

Cells isolated from fetal rat calvaria were cultured in a [14C]glycine-labeled collagen gel. This method of culture places the cells in an environment similar to that in the intact tissue and provides a means for assaying the release of collagenase from the cells. Degradation of the collagen matrix begins within the first 3 h of cell culture, the earliest time point at which samples were taken, and continues for at least 12 h. When cells were prepared from calvaria incubated for 30 min with parathyroid hormone (PTH), there was a marked decrease in the collagenase content of the freshly isolated cells, indicating that secretion of collagenase had been enhanced by PTH. Upon subsequent culture of control and PTH-pretreated cells, there was an increase in lysis of collagen gels surrounding the hormone-treated cells within 8 h. The increased collagenolysis was prevented by exposure of the cells to puromycin before and during culture, suggesting that collagenase synthesis also was stimulated by PTH.

Localization of Collagenase in Chronically Inflamed Guinea Pig Temporal Bone

Bone destruction, commonly associated with chronic otitis media, requires collagen degradation. Collagenase, a neutral protease, appears to be an essential component in the process of collagen breakdown. Collagenase was identified within chronically inflamed and normal guinea pig temporal bones using an immunohistochemical technique with fluorescein isothyocyanate and peroxidase-antiperoxidase labels. Localization of the enzyme identifies sites of matrix resorption. Collagenase was found in osteoclasts, osteocytes, mononuclear inflammatory cells, and at resorbing margins. Inflammation increased the intracellular collagenase content of inflammatory bone osteocytes when compared to normal osteocytes using a microspectrofluorometer. It appears that the inflammatory process directly influences bone destruction through the action of mononuclear inflammatory cells and indirectly by stimulating bone cells to increase their proteolytic enzyme production.

THE ROLE OF HUMAN SKIN COLLAGENASE IN EPIDERMOLYSIS BULLOSA

Human skin collagenase was quantitated by radioimmunoassay in 40 patients with various forms of epidermolysis bullosa to compare levels of the enzyme in blistered and clinically unaffected skin. Immunoreactive human skin collagenase was significantly elevated in the blistered skin of patients with both recessive and dominant forms of dystrophic epidermolysis bullosa (DEB). In addition, patients with generalized recessive DEB manifested a 4-fold increase in collagenase protein in normal-appearing skin, and patients with localized recessive DEB or epidermolysis bullosa letalis showed a 3-t to 3.5-fold elevation in the enzyme. However, patients with dominantly inherited DEB failed to displays a statistically significant increase in immunoreactive collagenase in nonblistered skin. Although it cannot be definitely stated whether the elevated collagenase content in the blistered skin represents a primary or secondary event, such as part of a wound healing response, the demonstration of markedly increased levels of collagenase in normal-appearing skin could, in part, provide an explanation at the molecular level for the formation of blisters in this disease.