Abstract
Detailed knowledge on the molecular and cellular mechanisms that control (re)-differentiation of vascular smooth muscle cells (SMCs) is critical to understanding the pathological processes underlying atherogenesis. We identified by differential display/reverse transcriptase-polymerase chain reaction 40 genes with altered expression in cultured SMCs upon stimulation with the conditioned medium of activated macrophages. This set of genes comprises 10 known genes and 30 novel genes, which we call "smags" (for smooth muscle activation-specific genes). To determine the in vivo significance of these (novel) genes in atherogenesis, we performed in situ hybridization experiments on vascular tissue. Specifically, FLICE (Fas-associated death domain-like interleukin-1beta-converting enzyme)-like inhibitory protein (FLIP) is expressed in neointimal SMCs as well as in lesion macrophages and endothelial cells, whereas the expression of the novel genes smag-63, smag-64, and smag-84 is restricted to neointimal SMCs. Characterization of full-length smag-64 cDNA revealed that it encodes a novel protein of 66 amino acids. smag-82 cDNA comprises the complete, unknown, 3'-untranslated region of fibroblast growth factor-5. Collectively, our results illustrate the complex changes of SMC gene expression that occur in response to stimulation with cytokines and growth factors secreted by activated macrophages. Moreover, we identified interesting candidate genes that may play a role in the differentiation of SMCs during atherogenesis.
Highlights
Detailed knowledge on the molecular and cellular mechanisms that control-differentiation of vascular smooth muscle cells (SMCs) is critical to understanding the pathological processes underlying atherogenesis
A substantial cellular part of the human atherosclerotic vessel wall consists of SMCs, the complexity and variability of these lesions, with respect to differences in cellular composition and differentiation status of those vascular cells, is too extensive to allow a direct comparison of gene expression
From these data we conclude that SMCs derived from this human, neonatal vascular tissue most closely resemble medial SMCs and are likely to exhibit the largest differences in gene expression upon activation, which makes them most appropriate for our studies
Summary
Human Tissue Samples—Arteries were dissected from human umbilical cords and immediately frozen or used for SMC explant cultures. As a control for equal RNA loading on the multiple tissue blots we used the 2-kb human -actin cDNA probe supplied with the filters. In situ hybridization was performed as described previously [10] For each probe both antisense and sense riboprobes were assayed, the latter ones as a control for specificity of the signal. Following an incubation for 10 min at 70 °C, second strand cDNA was synthesized for 1 h at 15 °C and 1 h at 21 °C by addition of 50 units of DNA Polymerase I and 4 units of RNase H (Life Technologies, Inc.) in 20 mM Tris (pH 7.5), 5 mM MgCl2, 100 mM KCl, 0.05 mg/ml bovine serum albumin, 10 mM dithiothreitol. In Vitro Transcription/Translation—After cloning full-length cDNAs into the pSP64 Poly(A) vector (Promega), in vitro transcription and translation was performed using the TnT Coupled Reticulocyte Lysate System (Promega)
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