Biomarkers of drug-induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury is an issue of concern because there are no obvious diagnostic markers for pre-clinical or clinical monitoring and there is an intellectual gap in our understanding of the pathogenesis of this lesion. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary targets, smooth muscle and endothelial cells are unknown. However, evaluation of novel markers for potential clinical monitoring with a mechanistic underpinning would add value in risk assessment and management. This mini review focuses on the progress to identify diagnostic markers of drug-induced vascular injury. Von Willebrand factor (vWF), released upon perturbation of endothelial cells, is transiently increased in plasma prior to morphological evidence of damage in dogs or rats treated with vascular toxicants. Therefore, vWF might be a predictive biomarker of vascular injury. However, vWF is not an appropriate biomarker of lesion progression or severity since levels return to baseline values when there is morphological evidence of injury. A potential mechanistically linked biomarker of vascular injury is caveolin-1. Expression of this protein, localized primarily to smooth muscle and endothelial cells, decreases with the onset of vascular damage. Since vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury.

Current status and future directions for diagnostic markers of drug-induced vascular injury.

Recently, there has been an increased incidence of vascular toxicity in pre-clinical toxicology studies. This is of concern because of the uncertain relevance and extrapolation of this finding to humans. In dogs, profound heart rate (HR) and mean arterial pressure (MAP) changes were considered surrogate markers for drug-induced vascular injury until the early 1990s when endothelin receptor antagonists (ETRA) did not significantly alter HR or MAP but induced identical lesions in the coronary arteries of dogs. Thus significant alterations in HR and MAP were found not to be a prerequisite for this lesion. Clinically, the potential for vascular injury coupled with the lack of an unequivocal non-invasive diagnostic marker is an issue of concern to pharmaceutical companies and the regulatory authorities. Therefore, qualification and validation of biomarkers as diagnostic tools for drug-induced vascular injury would add great value to risk management and expedite the drug development process. This review focuses on the status, progress and future trends in vascular biology aimed at identification and development of diagnostic markers that are specific, sensitive and possess potential utility in both a pre-clinical and clinical setting.

Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay.

BACKGROUND: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out. METHODS: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry. RESULTS: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells. CONCLUSIONS: The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures.

Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours.

Replication-incompetent recombinant retroviruses are currently used for gene delivery. The limited efficiency of gene transfer using these vectors hampers implementation of gene therapy. Successful integration of Moloney murine leukemia virus (MMuLV)-derived retroviral vectors into the host cell DNA requires cell division. The time difference between virus entry and cell division is variable and prolonged in slowly dividing cells. Therefore, the rate of intracellular decay of internalized vectors between the time of entry into the target cell and cell division may limit the probability of successful integration following viral entry. We present two methods that measure the intracellular stability of MMuLV-derived retroviral vectors in NIH 3T3 cells. The first is based on a temporary interruption of cell cycle progression by using cell detachment. This method provides an estimate, but not a direct measurement, of the half-life. The results show that the MMuLV intracellular half-life is on the order of but shorter than the total cell cycle time. The second method allows the direct measurement of the intracellular half-life by using two cell cycle-specific labels: 5-bromodeoxyuridine, a thymidine analog that labels cells in S-phase; and the viral vector that labels cells in mitosis. By varying the time between the administration of the two labels, the intracellular half-life is measured to be in the range of 5.5 to 7.5 h. Such a short intracellular half-life may restrict the efficiency of gene transfer by retroviral vectors, particularly in slowly dividing target cells.

Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors.

Clinical trials assessing the utility of cultured hematopoietic cells for the support of patients receiving high-dose chemotherapy are beginning. Although many reports have described these cultures, little is known about the donor-to-donor variability that might be expected to occur in widespread use. Therefore, this study was undertaken to assess variables which might predict and reduce the donor-to-donor variability in cell expansion potential. CD34-enriched cell cultures, plated to contain 3000 CD34+lin- cells per well, exhibited a wide range of cell output (0.02 to 5.07 x 10(6)) with a high coefficient of variation (CV = 0.69, n = 52). The range in CFU-GM output was even greater (12 to 9455, CV = 0.90). Addition of preformed stroma had a significant positive effect, and resulted in narrower ranges of cell (0.19 to 8.27 x 10(6), CV = 0.41) and CFU-GM (218 to 17586, CV = 0.54) output. A wide range of stromal-dependency was exhibited by CD34-enriched cells from different donors, with stroma augmenting cell output by 1.2- to 14-fold (mean 3.5), and CFU-GM output by 1.7- to 24-fold (mean 6.5). In contrast, changes in the soluble growth factor combination affected cells from different donors in a similar fashion, thereby altering the mean level of performance without reducing donor-to-donor variability. Experiments were next performed to assess the relative contribution of CD34+lin- cells and stromal cells to culture variability by culturing CD34+lin- cells from three donors on preformed stroma from three donors in parallel. Variability in culture output was attributed to the CD34+lin- cell donor, whereas stroma from different autologous or allogeneic donors gave similar performance. Therefore, both expansion potential and stromal-dependency were inherent characteristics of CD34+lin- cells from different donors. Donor characteristics (i.e., sex, age, weight, and height) and flow cytometric assays (i.e., CD34+lin- cell purity, and CD38-, Thy-1+, and c-kit+ subsets thereof) were not well correlated with expansion potential. In contrast, many of the different biological characteristics (i.e., inoculum CFU-GM, cell and CFU-GM output, and stromal-dependency) were strongly correlated with each other. Mononuclear cell (MNC) cultures, which provide an accessory cell environment (including endogenous stroma) in which CD34+lin- cells grow, were compared with CD34-enriched cell cultures. MNC cultures (containing 3000 CD34+lin- cells) were found to give the greatest and most consistent cell (2.51 to 5.20 x 10(6), CV = 0.17) and CFU-GM (2618 to 14,745, CV = 0.46) output. These results have significant implications for the design of clinical trials of cultured hematopoietic cells, as well as for the understanding of diversity in human stem cell behavior. Furthermore, the results demonstrate the importance of a large sample size in scientific studies of primary human hematopoietic cell behavior.

flt-3 ligand is more potent than c-kit ligand for the synergistic stimulation of ex vivo hematopoietic cell expansion.

The c-kit and flt-3 tyrosine kinase receptors are expressed on primitive hematopoietic cells, and ligands for both receptors have been cloned. In this study, the effects of c-kit ligand (KL) and flt-3 ligand (FL) were compared in the presence of IL-3, GM-CSF, and erythropoietin (3/GM/EPO), using frequent medium exchange cultures of human bone marrow mononuclear cells (BMMNC) and CD34-enriched cells. In MNC cultures, KL increased cell output by 1.7-fold (p < 10(-4), n = 13) and CFU-GM output by 2.4-fold (p < 10(-3)) as compared with control cultures containing only 3/GM/EPO. Analogously, FL increased cell output by 1.3-fold (p < 10(-3)) and CFU-GM output by 4.4-fold (p < 10(-6)). Therefore, FL was more potent on CFU-GM output than KL, but neither altered the lineage composition (granulocyte, monocyte, macrophage) of the colonies produced. Direct addition of KL or FL to colony assays resulted in only a 1.2-fold increase in CFU-GM outgrowth, suggesting that the effects on increased CFU-GM output were at the preprogenitor stage. In CD34-enriched cell cultures, the effects of KL and FL on CFU-GM output were similar (9-fold above control). Nevertheless, MNC cultures (containing an equivalent number of CD34+lin- cells) always generated more cells (2-fold to 4-fold) and CFU-GM (3-fold to 6-fold) than did parallel cultures of CD34-enriched cells. The greater effect of FL (over KL) in MNC cultures was probably due to synergy with endogenously produced growth factors that were absent in CD34-enriched cell cultures. FL-containing cultures (+/-KL) generated cells that formed larger colonies, and these cells had more proliferative potential on replating into secondary and tertiary cultures. Furthermore, FL increased the output of LTC-IC by 2.1-fold (p < 0.01) and CD34+lin- cells by 6-fold (p < 0.05) as compared with 3/GM/EPO cultures. In contrast, KL did not affect the output of LTC-IC and only slightly increased CD34+lin- cell output (by 1.4-fold). Erythrocytes were increased by KL (2.8-fold) and decreased by FL (0.6-fold), whereas granulocytes and monocytes were increased by both KL (1.4-fold) and FL (2.0-fold). When used together, KL and FL were completely additive with respect to cell, CFU-GM, and LTC-IC output, as well as lineage composition. The results indicate that FL is a more potent synergistic growth factor than KL for MNC expansion and that KL and FL act in an independent, direct, additive manner.

Immunofluorescence quantitation of stromelysin in human synovial fibroblasts by confocal laser scanning microscopy.

BACKGROUND: Elevated levels of stromelysin have been reported in humans with osteoarthritis and rheumatoid arthritis, as well as in animal models of arthritis. However, a considerable amount of heterogeneity is observed in the expression of this enzyme in pathologic tissues as well as in in vitro systems. To analyze this variability, stromelysin expression was quantitated in individual human synovial fibroblasts (HSF) obtained from osteoarthritis patients. EXPERIMENTAL DESIGN: HSF were incubated with interleukin-1 (40 units/ml), an agonist known to induce stromelysin, in the presence or absence of dexamethasone (0.01 to 100 nM), an inhibitor of stromelysin transcription. With a stromelysin-specific antibody and a tetramethyl-rhodamine 5-isothiocyanate-labeled secondary antibody, the enzyme was visualized and the fluorescence in individual cells was quantified with an ACAS 570 laser cytometer in confocal mode. RESULTS: Stromelysin expression varied from one cell to another; however, on the basis of the magnitude of expression of stromelysin by each cell, the "nonresponders" within each treatment were identified. Approximately 34% of the cells showed a higher level of stromelysin expression in IL-1-treated HSF compared with controls. A dose-dependent inhibition in the expression of stromelysin was observed in response to increasing concentrations of dexamethasone. The dose-dependent changes in the accumulation of stromelysin protein correlated well with the stromelysin mRNA expression. CONCLUSIONS: Confocal laser scanning microscopy can be effectively used to analyze cellular heterogeneity in stromelysin expression.

Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2-kinase in mouse T-lymphoma cells.

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 micrograms/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.

Flow cytometric characterization of lymphocyte subpopulations in the cynomolgus monkey (Macaca fascicularis).

Characterization of immune cell subpopulations in the cynomolgus monkey was performed using a direct immunofluorescence technique adaptable for routine and repeated monitoring. This whole blood procedure is faster and requires less volume than conventional density gradient isolation methods. Low intra- and inter-animal variations were seen in hematology parameters and in CD4, CD8, and CD20 lymphocyte subtypes. CD4 values were 28% of lymphocytes in males and 30% in females. Fifty-six percent were CD8+ in males and 54% in females. CD4:CD8 ratios were approximately 0.5 in both sexes. This proportion is the reverse of that observed in humans, but appears normal for the cynomolgus. Consistent with values reported for humans, approximately 12% of cynomolgus peripheral blood lymphocytes were CD20+. Greater than 95% of the lymphocytes present in blood were identified as CD4, CD8, or CD20 positive.

Comparative micronucleus quantitation in pre- and post-column fractionated mouse bone marrow by manual and flow methods.

In the present study, cellulose-column fractionation methodology which has been used to eliminate nucleated cells in bone marrow was verified for its usefulness in micronucleus analysis and compared to standard smear methodology using cyclophosphamide as the test compound. Also, the possibility of using column-fractionated cells in the evaluation of micronucleus frequency by flow cytometry has been explored and comparative results are reported. The results indicated that column fractionation was effective in removing nucleated cells from mouse bone marrow and provided clean preparations of polychromatic and normochromatic erythrocytes (PCEs and NCEs). An initial comparison of manual scoring of cyclophosphamide-induced (10, 20 or 40 mg/kg) micronucleus frequency between standard whole bone-marrow smear and column-fractionated cytospun smears from the same animals showed comparable results. In a definitive study, manual scoring of micronuclei in whole bone marrow was compared with the column-fractionated cell preparations quantified manually and using flow cytometry. Statistically significant positive dose-related trends were detected with all 3 methods, with each treatment group having significantly elevated micronucleated PCEs (MNPCEs) compared to the control group. The 3 methods provided comparable MNPCE values for the lower dose groups but diverged somewhat for the high dose group. The flow method yielded similar individual animal variability in the data when compared to the other two methods. These results support the use of column fractionation in the enumeration of MNPCEs and indicate that coupling this technique with flow cytometry may provide a rapid and sensitive method for the conduct of mouse bone-marrow micronucleus studies.

Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.

Regulation of human monocyte surface antigen expression. I. Up-modulation of Mo3e antigen expression on U-937 and HL-60 cells stimulated by pharmacologic activators of protein kinase C.

Mo3e is a protein (p 50,80) that is expressed on the surface of human monocytic cells after exposure in vitro to soluble activating factors that include bacterial lipopolysaccharide, muramyl dipeptide, and phorbol myristate acetate (PMA). The surface expression of Mo3e may represent a cellular event that occurs in response to the formation of "secondary messengers" that include diacylglycerol, inositol trisphosphate, and calcium ions. This postulate is based on the stimulatory effect of agents that can mimic the activity of endogenous diacylglycerol (PMA and other biologically active phorbol compounds, mezerein, and L-alpha-1,2 dioctanoylglycerol) and inositol trisphosphate (ionomycin) on Mo3e expression by U-937 and HL-60 cells. The inhibitory effect of phospholipid-active calmodulin inhibitors (trifluoperazine, chlorpromazine, and dibucaine), calcium antagonists (nicardipine and TMB-8), and EGTA further support the involvement of phospholipid- and calcium-dependent protein kinase (protein kinase C) and calcium ions in the up-modulation of Mo3e surface expression.

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e.

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.