Heparin/endothelial cell growth supplement regulates matrix gene expression and prolongs life span of vascular smooth muscle cells through modulation of interleukin-1.

Vascular smooth muscle cells produce and respond to interleukin-1, a cytokine which modifies inflammation-associated vascular activities including the synthesis of extracellular matrix proteins. We have established vascular smooth muscle cells culture conditions in which heparin, in the presence of endothelial cell growth supplement, promotes cell proliferation and inhibits interleukin-1 and matrix protein expression. To test whether interleukin-1 mediates growth and matrix modulation by heparin/endothelial cell growth supplement, vascular smooth muscle cells were transfected with an Epstein-Barr virus-derived expression vector designed to express interleukin-1 antisense transcripts. RNase protection and ELISA assays demonstrated a complete block of interleukin-1 transcription and protein synthesis. Northern blot analysis also showed that interleukin-1 antisense decreased the expression of matrix genes such as type I collagen, fibronectin, and decorin similar to downregulation after heparin/endothelial cell growth supplement treatment. In contrast, the expression of versican was not affected, indicating a selective suppression of matrix proteins. In addition, interleukin-1 antisense significantly prolonged the life span of vascular smooth muscle cells in culture. Our data suggest that heparin/endothelial cell growth supplement induces matrix remodeling and controls growth and senescence of vascular smooth muscle cells through down-regulation of interleukin-1.

Spontaneous p53 mutation in murine mesothelial cells: increased sensitivity to DNA damage induced by asbestos and ionizing radiation.

The p53 gene regulates the G1 cell cycle checkpoint in response to DNA damage. A primary murine mesothelial cell line (D9) spontaneously acquired a point mutation at codon 135 in exon 5 of the p53 gene, resulting in substitution of alanine for proline; early passage D9 cells expressed wild-type p53. The growth rate of late passage D9 cells that acquired the p53 mutation was increased compared to that of early passage cells; however, this mutation was not sufficient to confer tumorigenicity to this cell line. Mammalian cells that express wild-type p53 show a transient arrest in G1 after exposure to ionizing radiation. Early passage D9 cells showed a G1 arrest following ionizing radiation, while late passage D9 cells arrested in G2 or mitosis. The clastogenic effects of ionizing radiation can be demonstrated by the cytokinesis-arrested micronucleus assay. Following treatment with cytochalasin B to arrest cytokinesis, ionizing radiation induced micronuclei in 50% of late passage D9 cells compared to 15% of early passage cells. After exposure to 15 micrograms/cm2 of crocidolite asbestos fibers, 18% of late passage cells had micronuclei compared to 4% of early passage cells. It is hypothesized that loss of the G1 cell cycle checkpoint contributes to genetic instability in murine mesothelial cells.

Isolation and characterization of microvascular endothelial cells from the adult human dermis and from skin biopsies of patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a rheumatic autoimmune disease without known etiology and pathogenesis. Inflammatory processes with selective microvascular involvement seem to play an important role in the early stages of SSc. EXPERIMENTAL DESIGN: To elucidate the pathogenesis of the selective microvascular involvement in SSc, we have isolated microvascular endothelial cells from the adult human dermis (ADMEC) and for the first time from skin biopsies of patients with SSc (SSc-ADMEC) and established in cell culture. Ulex europaeus I-coated magnetic Dynabeads and the perfusion digestion technique were applied for endothelial cell isolation. RESULTS: The cultured ADMEC and SSc-ADMEC showed the endothelial cell-specific antigenic determinants of intracellular von Willebrand factor and platelet endothelial cell adhesion molecule-1 along their cell-cell borders. These cells displayed specific uptake of acetylated low-density lipoprotein. Normal ADMEC were additionally characterized for angiotensin I-converting enzyme activity. Tumor necrosis factor-alpha activated normal ADMEC and SSc-ADMEC expressed the inflammatory adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, similarly to tumor necrosis factor-alpha-activated large-vessel endothelium represented by human umbilical cord vein endothelial cells. Normal ADMEC and human umbilical cord vein endothelial cells also expressed beta 1- and beta 4-integrin receptors in cell culture. CONCLUSIONS: Normal ADMEC and SSc-ADMEC express endothelial cell-specific antigenic and biochemical determinants in vitro. SSc-ADMEC were for the first time established in cell culture.

Modulation of extracellular matrix gene expression by heparin and endothelial cell growth factor in human smooth muscle cells.

We have previously shown that human vascular smooth muscle cells grown for several passages in the presence of heparin and endothelial cell growth factor (ECGF) exhibit profound alterations in the synthesis of extracellular matrix proteins. In the present study, we demonstrate that the mRNA steady-state levels for various matrix macromolecules were altered in the presence of heparin and ECGF, but not ECGF alone. Specifically, the expression of types I and IV collagens, fibronectin, and decorin proteoglycan genes were markedly inhibited, whereas that of versican proteoglycan and beta-actin genes were unaffected. The effects were fully reversible. The suppression of the collagen gene expression was related to decreased collagen production by cells incubated with heparin and ECGF. Thus, heparin and ECGF synergistically elicit a coordinate and selective inhibition of matrix gene expression in human smooth muscle cells.