Phenotypic Heterogeneity Influences Apoptotic Susceptibility to Retinoic Acid and cis -Platinum of Rat Arterial Smooth Muscle Cells In Vitro

Abstract

Abstract-Rat aortic smooth muscle cells (SMCs) cultured from intimal thickening 15 days after endothelial injury (IT-15), unlike those of normal media, show a monolayered, epithelioid phenotype and high levels of cellular retinol binding protein-1 (CRBP). Epithelioid clones obtained from the normal media suggest a "mosaicism" of arterial SMCs. Intimal cell homeostasis from the balance of proliferation and apoptosis is critical for the progression of vascular lesions. All-trans retinoic acid (tRA) reduced [ 3 H]thymidine incorporation and G 1 3 S phase progression of IT-15 and epithelioid clone but not of normal media and IT 60 days after injury (IT-60) SMCs. Hoechst staining, flow cytometry, and ligation-mediated polymerase chain reaction showed an increased susceptibility of IT-15 and epithelioid clone to tRA and cis-diaminedichloroplatinum II (CDDP)-induced apoptosis and cytotoxicity compared with normal media and IT-60 cells. The latter retained an increased susceptibility to tRA-induced apoptosis compared with normal media SMCs. tRA-induced apoptosis associated with an increased ratio of bax to bcl-2 by bax overexpression and cleavage of caspase-3. Anti-CRBP but not anti-IgG antibody prevented tRA-induced apoptosis and changes in related signaling molecules but not CDDP effects. Our findings support the relevant role of phenotypic heterogeneity in the determining proliferative as well as apoptotic behavior of arterial SMCs.

Propionyl-L-carnitine Reduces Proliferation and Potentiates Bax-related Apoptosis of Aortic Intimal Smooth Muscle Cells by Modulating Nuclear Factor-κB Activity

“…Cell viability was calculated using 0.4% trypan blue exclusion in triplicate. [ 3 H]Thymidine incorporation was determined as previously reported (32).…”

“…For immunofluorescence staining in vitro, cells growing on glass slides were fixed for 5 min in cold methanol (Ϫ20°C), rinsed twice in phosphate-buffered saline, and incubated with anti ␣-actin, anti-Bax, anti-p65, and anti-p50 monoclonal or polyclonal antibodies (32). Cells were photographed using a Nikon fluorescent microscope and DNA chromatin morphology under UV visualization.…”

“…Cells were photographed using a Nikon fluorescent microscope and DNA chromatin morphology under UV visualization. Apoptosis of adherent cells was investigated by calculating the percentage of nuclei showing apoptotic features using Hoechst staining in triplicate (32). Assigning a serial number to each slide ensured the objectivity of all measurements.…”

“…Flow Cytometry and Cell Cycle Analysis-Flow cytometry was performed as previously reported using a fluorescence-activated cell sorting scan flow cytometer (BD Biosciences) and a Lysis II program (32). The number of cells in the sub-G 1 (DNA content Ͻ2N), G 0 /G 1 , S, and G 2 /M phases was expressed as a percentage of total events (10,000 cells).…”

“…DNA Isolation and Ligation-mediated PCR-In order to better identify and quantify apoptosis-associated DNA fragmentation, DNA was extracted, and ligation-mediated PCR was performed (32). The nucleosomal ladder was quantified in 1.2% agarose gels stained with ethidium bromide (32).…”