Dennis A. Laska

Characterization of metalloprotease cleavage products of human articular cartilage

OBJECTIVE To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage. METHODS Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry. RESULTS Complete sequences of the peptides proteolyzed from human articular cartilage, including N- and C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases. CONCLUSION The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.

A prospective study of multiple protein biomarkers to predict progression in diabetic chronic kidney disease

BACKGROUND Diabetic nephropathy imposes a substantial cardiovascular and renal burden contributing to both morbidity and excess mortality. Progression of chronic kidney disease (CKD) in diabetes mellitus is variable, and few biomarkers are available to predict progression accurately. Identification of novel predictive biomarkers may inform clinical care and assist in the design of clinical trials. We hypothesized that urinary and plasma protein biomarkers predict CKD progression independently of the known clinical markers such as albuminuria and estimated glomerular filtration rate (eGFR) in diabetic nephropathy. METHODS We studied 67 US veterans with CKD due to type 2 diabetes mellitus and 20 age-matched controls (no CKD, hypertension or cardiovascular disease). After clinical evaluation and the collection of blood and urine specimens for 24 biomarkers, we followed subjects prospectively for the next 2-6 years. CKD progression was defined in three ways: (i) clinically by examining eGFR versus time plots for each individual (slope progression), (ii) progression to end-stage renal disease (ESRD) and (iii) a composite outcome of ESRD or death. RESULTS Among 17 urinary and 7 plasma biomarkers evaluated, the relationship of the biomarkers with outcome was as follows: (i) for progression identified by eGFR plots, urinary C-terminal fibroblast growth factor (FGF)-23 emerged to have the strongest primary association (adjusted odds ratio [aOR] 2.08, P = 0.008); (ii) for ESRD, plasma vascular endothelial growth factor (VEGF) had an association (aOR: 1.44, P = 0.027) and (iii) for the composite outcome of death and ESRD, plasma C-terminal FGF-23 also had a robust direct association (aOR: 3.07, P = 0.008). CONCLUSION The relationship of biomarkers with future progression of CKD is complex and depends in part on how CKD progression is defined. Biomarkers in the FGF-23 and VEGF-A pathways predicted patient progression independently of albuminuria levels in this patient cohort. Additional studies in other cohorts will help further validate this pilot study.

Evaluation of an in vitro coculture model for the blood-brain barrier: comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources.

SummaryCocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.

Characterization and application of a vinblastine-selected Caco-2 cell line for evaluation of P-glycoprotein

SummaryThe role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to upregulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Comparison of in vitro and in vivo models to assess venous irritation of parenteral antibiotics

The venous irritation potential of four parenteral antibiotics, tetracycline hydrochloride (TET), erythromycin lactobionate (ERY), amphotericin B (AMP), and cephaloridine (CEP), was evaluated in an in vivo model using the rabbit ear vein. Lateral ear veins of New Zealand White rabbits were infused for 1 hr with test solutions containing TET (0.25,2.5, or 10 mg/ml), ERY (2.5, 5, or 25 mg/ml), AMP (0.05, 0.1, or 0.5 mg/ml), or CEP (4 or 20 mg/ml). Control rabbits received comparable volumes of 0.9% NaCl or 5% dextrose. Approximately 24 hr postinfusion, the rabbits were evaluated for visually evident changes in the treated ears. Pathologic evaluation of the veins was performed using histologic sections and scanning electron microscopy. TET, ERY, and AMP caused concentration-dependent changes in veins characterized primarily by loss of endothelium with associated inflammation and thrombus formation, consistent with the known clinical irritancy of these antibiotics. CEP, on the other hand, was well tolerated in the rabbit ear vein, paralleling its low irritancy potential in man. Test solutions identical to those used in vivo in rabbits were also evaluated in established in vitro assays for hemolytic potential when mixed with whole blood from monkeys and for damage to L6 muscle cells as determined by loss of creatine phosphokinase. Results of the in vitro test systems paralleled those of the rabbit ear model, with TET, ERY, and AMP exhibiting dose-dependent hemolysis and muscle cell toxicity, while CEP was comparatively nontoxic. Of the three models, the rabbit ear vein had the greatest sensitivity when histopathologic evaluation was employed.(ABSTRACT TRUNCATED AT 250 WORDS)

Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1)

Rapid intravenous adminstration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30–60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-releated degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to relase histamine and to study mechanisms of histamine release than RBL-1 cells.

In Vitro and in Vivo Ultrastructural Changes Induced by Macrolide Antibiotic LY281389

High doses of LY281389 (9-N-(n-propyl)-erythromycylamine) cause cytoplasmic vacuolar changes in striated and smooth muscle characteristic of drug-induced phospholipidosis. This study characterized phospholipidosis in striated and smooth muscle of rats and dogs, compared in vivo observations with those in a cultured rat myoblast model, and attempted to confirm the lysosomal origin of the drug-induced vacuoles. Standard transmission electron microscopy and acid phosphatase cytochemistry techniques were used to evaluate ultrastructural changes in vivo and in vitro. Rats and dogs exposed to LY281389 had a time- and dose-related increase in number and size of vacuoles containing concentric lamellar figures in cardiac and skeletal muscle. Cytochemical staining of dog stomach smooth muscle for acid phosphatase, a lysosomal enzyme, stained the periphery of vacuoles that contained concentric lamellar figures. Cultured rat L6 myoblast cells were exposed to 0.25 mg LY281389/ml for 2.5, 5, 10, 20, 30, or 90 min and 2, 6, 12, 24, or 48 hr. Cell cultures exposed for 2 hr had several predominantly large, clear, membrane-bound vacuoles, and at 6 and 12 hr there were greater numbers of large vacuoles that contained increased amounts of membranous figures. Following 24- or 48-hr exposures, vacuoles occupied most of the cytoplasmic volume, and were engorged predominantly with amorphous or granular material. These findings indicate that LY281389 can induce similar phospholipidosis-like vacuolar changes in rat and dog muscle and in a cultured rat muscle cell line. Further, positive acid phosphatase staining of drug-induced vacuolar structures, in conjunction with standard transmission electron microscopy techniques, strongly suggests that vacuoles seen in vitro and in vivo are lysosomal in origin.

In vitro correlation of ultrastructural morphology and creatine phosphokinase release in L6 skeletal muscle cells after exposure to parenteral antibiotics

SummaryMorphologic changes in a rat skeletal muscle cell line (L6) exposed for 1 h to the parenteral antibiotics amphotericin B (AMP), tetracycline-HCl (TET), erythromycin lactobionate (ERY), and cephaloridine (CEP) were characterized by transmission and scanning electron microscopy and compared to cellular release of creatine phosphokinase (CRK). AMP (0.05, 0.1, 0.5 mg/ml) caused a concentration-related swelling of nuclei, endoplasmic reticulum, and mitochondria. Loss of membrane integrity associated with AMP exposure was evident at the middle concentration and extensive at the high concentration, which correlated well with the 43 and 90% depletion of CPK from the muscle cells, respectively. TET (0.25, 1.0, 2.5 mg/ml) caused dilation of endoplasmic reticulum and cytoplasmic blebbing at the low concentration but had no effect on the cytoplasmic membrane or CPK. Cells exposed to the high concentration of TET had extensive damage to the cytoplasmic membrane, and CPK was completely depleted. ERY (2.5, 5.0, 25 mg/ml) caused a pattern of morphologic changes and CPK depletion similar to TET. CEP (4.0, 20, 50 mg/ml) had no effect on membrane integrity or CPK; however, membranous whorls were prominent in the cytoplasm. A good correlation between CPK release and cytoplasmic membrane integrity was evident and the ability of these agents to release CPK from muscle cells in culture correlated with the known irritancy potential of these parenteral antibiotics. Furthermore, CPK depletion seems to be a reliable indicator of muscle cell damage after cytoplasmic membrane perturbation and is therefore an appropriate index of toxicity in this in vitro muscle irritation model.

Differential Toxicity of Parenteral Antibiotic Drugs in Renal Cells (LLC-PK1) Grown on Permeable Membrane Filters

Growth of renal epithelial cells on permeable membrane niters promotes the expression of polarized function and facilitates the study of directional aspects of exposure of kidney cells to nephrotoxins (apical versus basolateral). Monolayer cultures of LLC-PK1 cells were grown on membrane filter inserts coated with a collagen-laminin matrix and incubated with three parenteral antibiotic drugs (vancomycin, 2.5–15 mg/ml; tobramycin, 0.5–10 mg/ml; and cephaloridine, 0.05–0.5 mg/ml) at 37d`C for 48 h. Growth medium containing the experimental antibiotic drugs was placed in the upper or lower chamber for apical or basolateral exposure, respectively. After incubation, cellular viability was determined by nigrosin dye exclusion and by the in situ uptake of the fluorescent dye, propidium iodide, which is increased in dead cells. Cytotoxicity was seen with increasing concentrations of each antibiotic drug after basolateral or apical exposure. Profound basolateral and apical differences, expressed as a percent of co...

The Agar Diffusion Cytolysis Method: An Alternative In Vitro Screen for the Prediction of a Severe Ocular Response

Manufacturing intermediates and formulation components constitute the bulk of compounds evaluated in ocular irritation protocols. A cost-effective in vitro screen for predicting severe ocular reactions, which reduces the chance of exposing animals to caustic chemicals, was a logical first step in a tiered approach to the reduction, refinement, and eventual replacement of animals in ocular irritation test protocols. Our laboratory has validated the agar diffusion cytolysis method, which employs a monolayered SIRC cell culture as an initial biological ocular screen. Twenty-four hours after plating 100-mm dishes, the culture medium was replaced with a 0.01% neutral red/1% agar overlay and incubated for a minimum of 16 h. Solid or liquid compounds were applied atop the agar diffusion matrix in glass cylinders or on paper discs, respectively, at doses of 2, 5, 10, and 25 mg or μ1 and incubated at 37°C for 24 h. Clear zones resulting from the loss of the neutral red dye following compound-induced cytolysis were...