A new method for continual quantitation of viable cells on endothelialized polyurethanes

Nikolaychik, Victor; Samet, M. M.; Lelkes, Peter I.

doi:10.1163/156856296x00057

Cited by 48 publications

(25 citation statements)

References 23 publications

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“…Both compounds were able to inhibit MLV infection but less effectively than Flavo (Ros IC 50 ϭ 13.7 M, DRB IC 50 ϭ 15.4 M; data not shown). Since CDKIs induce apoptosis in numerous cell types, we next examined the CDKIs' effect on cell viability by performing Alamar blue proliferation assays (36). No significant toxic effects were detected when concentrations of Flavo were Յ300 nM and when concentrations of Purv and MeO-Ros were Յ10 M (data not shown).…”

Section: Cdkis Block Moloney MLV Infectionmentioning

confidence: 99%

Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

Chao

Walker

Chanda

et al. 2003

Molecular and Cellular Biology

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Cyclin-dependent kinase inhibitors (CDKIs) have been shown to block human immunodeficiency virus and herpes simplex virus. It is hypothesized that CDKIs block viral replication by inhibiting transcription of specific cellular genes. Here we find that three CDKIs, flavopiridol, purvalanol A, and methoxy-roscovitine, block Moloney murine leukemia virus (MLV) transcription events. Using gene expression microarray technology to examine the inhibitory effects of CDKIs, we observed a cellular gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, down-regulated by CDKI treatment. The PBX consensus element (PCE), TGATTGAC, is conserved in the long terminal repeats of several murine retroviruses, including Moloney MLV. Mutations in the PCE completely inhibited viral transcription whereas overexpression of PBX1 and a PBX1-associated protein, PREP1, enhanced viral transcription. The interaction between the PCE and PBX1-PREP1 proteins was confirmed by gel shift experiments. Blocking PBX1 protein synthesis resulted in a significant decrease in viral transcription. Collectively, our results represent the first work demonstrating that the homeodomain proteins PBX1 and PREP1 are cellular factors involved in Moloney MLV transcription regulation.Cyclin-dependent kinases (CDKs) are key regulators of cell cycle control and transcription. Nine CDKs have been identified thus far. CDK1 to CDK7 are required for cell cycle regulation, and CDK7 to CDK9 are involved in RNA polymerase II-dependent transcription (33). In fact, CDK2, CDK4, and CDK6 also regulate transcription by activating E2F via specific phosphorylation of the retinoblastoma gene product (Rb protein) in complex with E2F (13, 49). It has been shown elsewhere that approximately 90% of all neoplasias are associated with CDK hyperactivation and subsequent Rb pathway inactivation (21). Therefore, CDKs represent attractive targets for cancer therapy, and several CDK inhibitors (CDKIs) have been developed (4,16,18,48). Two CDKIs, flavopiridol (Flavo) and UCN-1, are already in clinical trials as potential anticancer therapeutics (44,49).The antiviral activities of CDKIs were first observed in studies describing their interaction with positive transcription elongation factor b (P-TEFb), a factor required for human immunodeficiency virus (HIV) replication (32, 54). P-TEFb, composed of CDK9 and a cyclin subunit derived from one of three different genes (cyclin T1, T2, or K), controls RNA polymerase II-dependent transcription elongation by phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (41).

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Section: Cdkis Block Moloney MLV Infectionmentioning

confidence: 99%

Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

Chao

Walker

Chanda

et al. 2003

Molecular and Cellular Biology

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“…19 Alamar Blue indicator was added to the tissue culture well at a ratio of 200 L indicator to 2 mL of media. The plates were then incubated for 2 h at 37°C.…”

Section: Alamar Blue Assaymentioning

confidence: 99%

In vitro cytotoxicity andin vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) hydrogels

Suggs

Shive

García

et al. 1999

J. Biomed. Mater. Res.

121

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The in vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were assessed in order to investigate the influence of poly(ethylene glycol) molecular weight and copolymer composition. These materials have application as injectable cardiovascular implants; cytotoxicity due to leachable products, as well as inflammation caused by the biomaterial itself, may ultimately affect the biocompatibility of the implant. We utilized a 7-day in vitro cytotoxicity assay to quantify cell density and cellular proliferation in the presence of copolymer films. The copolymer films exhibited slight to moderate cytotoxicity toward cultured endothelial cells, showing 20-86% viability relative to controls. Cell viability increased with an increasing weight percent of PEG or, to a lesser extent, the molecular weight of PEG. In vivo biocompatibility was assessed using a cage implantation model over a 21-day time period. This system was used to characterize the local cellular and humoral inflammatory response in the surrounding exudate, as well as the size and density of macrophages adherent to the material itself. All copolymer formulations exhibited excellent biocompatibility relative to controls with no significant differences in total leukocyte count among the different formulations. The in vivo inflammatory reaction displayed normal wound healing over 21 days as shown by a progressive decrease in both leukocyte concentration and enzymatic activity. The surface coverage of the copolymer films remained relatively constant from 7 to 21 days. There were no cells larger than 0.003 mm2, which was previously shown to be the threshold value for foreign-body giant cells. These data suggest that P(PF-co-EG) hydrogels have potential for use as injectable biomaterials.

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“…23,24,37 Furthermore, the insoluble, intracellular crystals that disrupt the cellular membrane result in an extracellular precipitate attached to the polymeric substrate that on polyurethanes remains bound to the substrate even after extraction by detergent, thus affecting the outcome of this particular assay. 38 Deturgescense has been used to study endothelial viability in vitro, but its usefulness is limited because of its low sensitivity, 39 whereas the measurement of transendothelial cell potential, that is, cell pump function, destroys the sample. 40 [ 3 H]-thymidine incorporation has been used as a measure of viability and cell proliferation in EC, 41 but radioisotopes have many disadvantages, including the terminal nature of the measurement, labor-intensive handling and disposal (along with expense), and excessive processing time with 3 H-thymidine incorporation.…”

Section: Discussionmentioning

confidence: 99%

A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses

Seifalian¹,

Salacinski²,

Punshon³

et al. 2001

J. Biomed. Mater. Res.

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For the improvement of vascular graft patency, an endothelial cell (EC) lining is desirable. It is essential that the EC remains viable after being seeded onto the prosthetic graft. The aim of this study was to adapt an Alamar redox assay (ABRA) as a technique to monitor the viability of ECs seeded on prosthetic grafts. To test the graft types, we seeded human umbilical vein ECs on compliant polyurethane (CPU), expanded polytetrafluoroethylene, and Dacron at a density of 2 x 10(5) cell/cm(2). After 24 h of incubation, ABRA was added, and the absorbance was measured at 4, 8, and 24 h. To assess seeded cell concentrations on grafts, we seeded CPU at densities ranging from 1 x 10(5) to 8 x 10(5) cell/cm(2). The validity of the test was assessed with sodium azide and mitomycin C, known physiological perturbators. ABRA reduction demonstrated that ECs were viable and functional postseeding on the prosthetic grafts. A significant correlation was observed with ABRA reduction and cell concentrations (p < 0.001). The acid phosphatase assay demonstrated enzyme activity in the cells, but they were not maintained under normal physiological conditions. The ABRA bioreduced product was soluble, stable, and noncytotoxic over 24 h. The assay is independent of the geometry or physiochemistry of the graft type. The technique allows the continuous assessment of the metabolism and viability of seeded cells, is simple to perform, and does not destroy the cells or graft materials.

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A new method for continual quantitation of viable cells on endothelialized polyurethanes

Cited by 48 publications

References 23 publications

Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

In vitro cytotoxicity andin vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) hydrogels

A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses

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