Determination of the number of endothelial cells in culture using an acid phosphatase assay

Connolly, Daniel T.; Knight, Mary Beth; Harakas, Nikos K.; Wittwer, Arthur J.; Feder, Joseph

doi:10.1016/0003-2697(86)90131-4

Cited by 200 publications

(116 citation statements)

References 8 publications

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“…After washing to remove non-adherent cells, adherent cells were quantified using the acid phosphatase assay by incubation with the substrate solution (0.1 M sodium acetate, pH 5.5, 10 mM p-nitrophenylphosphate, and 0.1% Triton X-100; 100 l/well) for 2 h at 37°C (29). The reaction was stopped by the addition of 15 l of 1 N NaOH/well, and A 450 was measured.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Novel Integrin αMβ2 Binding Site in CCN1 (CYR61), a Matricellular Protein Expressed in Healing Wounds and Atherosclerotic Lesions

Schober

Lau

Ugarova

et al. 2003

Journal of Biological Chemistry

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CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediateearly gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin ␣ M ␤ 2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the ␣ M I domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SS-VKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-␣ M monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the ␣ M I domain (GST-␣ M I) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or ␣ M I domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates ␣ M ␤ 2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-␣ M I binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin ␣ M ␤ 2 binding motif that bears no apparent homology to any ␣ M ␤ 2 binding sequence reported to date. CCN11 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF), products of immediate-early genes transcriptionally activated in fibroblasts and smooth muscle cells upon growth factor stimulation, belong to the CCN (CYR61/CTGF/nephroblastoma-overexpressed) family of matricellular signaling molecules that have been implicated in diverse biological functions (1-3). Other CCN family members include CCN3 (nephroblastoma-overexpressed, Nov) and the wnt-1-induced secreted proteins CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). These conserved and modular proteins consist of an N-terminal secretory peptide followed by four structural domains that include 1) an insulin-like growth factor-binding protein homology domain, 2) a von Willebrand factor type C domain, 3) a thrombospondin type 1 repeat homology domain, and 4) a C-terminal domain with heparin binding motifs and sequence similarity to the C termini of von Willebrand factor and mucin (4). Consistent with their localization to the extracellular matrix and their homology to extracellular matrix proteins, several CCN members have been shown to support cell adhesion, induce adhesive signaling, promote cell migration, and augment growth factor-induced cell proliferation through interaction with integrin receptors and cell surface heparan sulfate proteoglycans (HSPGs) (5-13). Furthermore, both CCN1 and CCN2 induce neovascularization in the rat corneal micropocket assay (8, 9). Recentl...

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Section: Methodsmentioning

confidence: 99%

Identification of a Novel Integrin αMβ2 Binding Site in CCN1 (CYR61), a Matricellular Protein Expressed in Healing Wounds and Atherosclerotic Lesions

Schober

Lau

Ugarova

et al. 2003

Journal of Biological Chemistry

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“…The effect of rPD-ECGF or E. coli TP addition on endothelial cell proliferation was determined over an 8-day period by the acid phosphatase assay for endothelial cell number (Connolly et al, 1986). Calf pulmonary artery endothelial (CPAFi) cells were maintained as previously described (Finnis et al, 1992).…”

Section: Endothelial Cell Proliferation Assaymentioning

confidence: 99%

“…The mitogenic ,properties of rPD-ECGF toward endothelial cells was investigated by measuring cell proliferation. Cell number was measured indirectly over an 8-day period by an acid phosphatase enzyme assay (Connolly et al, 1986). CPAE cells were chosen as the target cell type because of their less stringent serum requirement compared to HUVE cells.…”

Section: Mitogenieity Of Pd-ecgfmentioning

confidence: 99%

Thymidine phosphorylase activity of platelet‐derived endothelial cell growth factor is responsible for endothelial cell mitogenicity

Finnis

Dodsworth

Pollitt

et al. 1993

European Journal of Biochemistry

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Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98 % purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; K,, 0.11 mM; V, , , 12.5 mmol min-' mg-'; turnover number, 9.4 s -I ) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

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“…Figure 1 shows cell viability estimated by measuring acid phosphatase activity, which is an accurate indicator of the number of endothelial cells. 19) No significant difference in cell viability was detected between untreated and BSO-pretreated cells. In BAECs that were not pretreated with BSO, the viability after 24-h exposure to 1 mM MG was decreased by 28% compared to the control.…”

Section: Effect Of Bso On Viability Of Baecs Exposed To Mgmentioning

confidence: 88%

“…The assay for acid phosphatase activity, which is an accurate indicator of the number of endothelial cells in culture, was accomplished using the method of Connolly et al 19) BAECs or HAECs (3ϫ10 4 cells) on 96-well plates were pretreated with BSO for 16 h and exposed to MG for 24 h. Then, the medium containing detached cells was removed. Cells remaining on the 96-well plates were washed with DPBS and incubated with 100 ml of 0.1 M 556…”

Section: Methodsmentioning

confidence: 99%

Buthionine Sulfoximine Promotes Methylglyoxal-Induced Apoptotic Cell Death and Oxidative Stress in Endothelial Cells

Takahashi

Tatsunami

Oba

et al. 2010

Biological & Pharmaceutical Bulletin

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Methylglyoxal (MG) is a reactive dicarbonyl compound that is formed as a metabolic byproduct of glycolysis. 1,2) MG is found at high levels in the blood of diabetic patients. 3) MG causes nonenzymatic glycation of proteins to yield irreversible advanced glycation end products (AGEs), leading to cross-linking or degradation of proteins. 4) MG has been linked to the development of diabetic complications related to vascular injury. [3][4][5] MG might contribute to not only the development of diabetes but also the pathogenesis of hypertension. 6) MG induces the apoptosis of Schwann cells, 7,8) human monocytic leukemia U937 cells, 9) and human umbilical vein endothelial cells. 10) It is suggested that oxidative stress is involved in MG-induced apoptosis. Oxidative stress resulting from the accumulation of reactive oxygen species (ROS) has been well characterized in diabetic complications. 11,12) MG inactivates antioxidant enzymes, such as glutathione peroxidase and Cu,Zn-superoxide dismutase. 13,14) The dysfunction of antioxidant defense systems may lead to the progression of oxidative stress and finally cell death, such as apoptosis.Reduced glutathione (GSH), the most abundant non-protein thiol antioxidant in cells, is important for protection against oxidative injury. Chronic intake of MG by mice caused the depletion of blood GSH. 15) Intracellular GSH was depleted in Schwann cells incubated with MG. 8) It was also revealed that N-acetyl-L-cysteine, an antioxidant, restored the MG-induced GSH depletion in Schwann cells and prevented apoptosis. In human monocytic leukemia U937 cells, MG-induced apoptosis and oxidative stress were enhanced by DLbuthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, and partially blocked by N-acetyl-L-cysteine. 9) However, it remains unknown whether BSO or N-acetyl-Lcysteine affects MG-induced apoptosis and oxidative stress in endothelial cells. Recently, we reported that MG induces apoptosis and oxidative stress in bovine aortic endothelial cells (BAECs). 16,17) MG affected GSH levels in BAECs. 17) In this study, we examined the effect of BSO on MG-induced apoptosis and oxidative stress by using BAECs. MATERIALS AND METHODSMaterials MG (40% aqueous solution), BSO, and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS) was from Lonza (Walkersville, MD, U.S.A.). Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), phosphate-buffered saline (PBS) at pH 7.4, Lglutamine, penicillin, and streptomycin were from Gibco BRL (Grand Island, NY, U.S.A.). All other chemicals used were of reagent grade.Endothelial Cell Culture BAECs were purchased from Lonza. Cells were obtained at the third passage, transferred to 75 cm 2 filter vent flasks, and grown to 80-90% confluence in DMEM containing 10% FBS, 4 nM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, with incubation at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. Cells were passaged by trypsinization and those b...

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Determination of the number of endothelial cells in culture using an acid phosphatase assay

Cited by 200 publications

References 8 publications

Identification of a Novel Integrin αMβ2 Binding Site in CCN1 (CYR61), a Matricellular Protein Expressed in Healing Wounds and Atherosclerotic Lesions

Identification of a Novel Integrin αMβ2 Binding Site in CCN1 (CYR61), a Matricellular Protein Expressed in Healing Wounds and Atherosclerotic Lesions

Thymidine phosphorylase activity of platelet‐derived endothelial cell growth factor is responsible for endothelial cell mitogenicity

Buthionine Sulfoximine Promotes Methylglyoxal-Induced Apoptotic Cell Death and Oxidative Stress in Endothelial Cells

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