Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system.

Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.

Thy-1, a novel marker for angiogenesis upregulated by inflammatory cytokines.

We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti-Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1-Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.

APEG-1, a novel gene preferentially expressed in aortic smooth muscle cells, is down-regulated by vascular injury.

Despite the importance of phenotypic alterations in arterial smooth muscle cells (ASMC) during the pathogenesis of arteriosclerosis, little is known about genes that define differentiated ASMC. Using differential mRNA display, we isolated a novel gene preferentially expressed in the rat aorta and termed this gene APEG-1. The cDNA of rat APEG-1 contained an open reading frame encoding 113 amino acids, which would predict a basic protein of 12.7 kDa. The amino acid sequence of rat APEG-1 was highly conserved among human and mouse homologues (97 and 98%, respectively). Using an APEG-1 fusion protein containing an N-terminal c-Myc tag, we identified APEG-1 as a nuclear protein. By in situ hybridization, APEG-1 mRNA was expressed in rat ASMC. Although APEG-1 was expressed highly in differentiated ASMC in vivo, its expression was quickly down-regulated and disappeared in dedifferentiated ASMC in culture. In vivo, APEG-1 mRNA levels decreased by more than 80% in response to vascular injury as ASMC changed from a quiescent to a proliferative phenotype. Taken together, these data indicate that APEG-1 is a novel marker for differentiated ASMC and may have a role in regulating growth and differentiation of this cell type.

Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells.

Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation.

Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

CD44, the principal receptor for hyaluronic acid, is a widely distributed cell surface proteoglycan involved in cellular activation, proliferation, and migration. These processes are also central to the vascular smooth muscle cell's response to arterial wall injury. We evaluated the expression of CD44 and its isoform, CD44-V6, on vascular smooth muscle cells in vitro and in vivo and assessed the role of CD44 in DNA synthesis. Cultured vascular smooth muscle cells expressed CD44 and CD44-V6 at levels equal to or higher than those of the beta 1 and beta 2 integrins. In a rat carotid artery balloon injury model, CD44 and CD44-V6 mRNAs were unregulated in vascular smooth muscle cells after injury, and CD44 protein expression was greatest at the luminal edge of the growing neointima. CD44-expressing smooth muscle cells proliferated actively, and hyaluronic acid expression increased after injury in a temporal pattern similar to that of CD44. Through binding to hyaluronic acid, CD44 augmented DNA synthesis in cultured human and rat smooth muscle cells by 48 +/- 7.8 and 100 +/- 12.5%, respectively, an effect inhibited by an anti-CD44 antibody that blocked hyaluronate binding. These observations support a role for CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.