Hiroshi Wachi

Increase in Elastin Gene Expression and Protein Synthesis in Arterial Smooth Muscle Cells Derived From Patients with Moyamoya Disease

BACKGROUND AND PURPOSEnMoyamoya disease is a progressive cerebrovascular occlusive disease that is rare in all ages but frequently presents in children. The etiology of the disease is unknown. We examined elastin gene transcripts and elastin synthesis in cultured arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those in SMCs from age-matched control subjects.nnnMETHODSnWe used six cell strains from moyamoya patients and four from controls. The expression of elastin protein was observed by Western blot analysis and metabolic labeling with 3H-valine. Elastin gene transcripts were identified by Northern blot analysis.nnnRESULTSnElastin mRNA and protein levels were elevated in all SMCs from moyamoya patients compared with control SMCs. Although transforming growth factor-beta 1 (TGF-beta 1), a potent enhancer of the expression of elastin in arterial SMCs, upregulated elastin mRNA and protein levels in SMCs from both moyamoya patients and control subjects, the maximum levels of elastin synthesis and elastin gene transcripts in response to exogenous TGF-beta 1 were significantly greater in moyamoya SMCs than control SMCs. In addition, quiescent moyamoya SMCs secreted significantly more TGF-beta 1 into the culture medium than quiescent control SMCs (P < .01).nnnCONCLUSIONSnOur findings suggest that moyamoya disease may result, at least in part, from an abnormal regulation of extracellular matrix metabolism that leads to increased steady state levels of elastin mRNA and elastin accumulation in the intimal thickening and that increased elastin accumulation is a stable marker of SMCs from patients with moyamoya disease.

Modulation by elastin peptide VGVAPG of cell proliferation and elastin expression in human skin fibroblasts

Elastin is a major component of dermal connective tissue and confers elasticity to the tissues [1]. Elastin has unique repeating sequences in the hydrophobic region: the pentapeptide VPGVG and the hexapeptide XPGVGV (X = 1 or V). The pentapeptide VPGVG is the only repeating sequence present in the elastin molecules of all animal species so far analyzed including human, bovine, porcine and chicken elastin [2–6]. VGVAPG is a hexapeptide repeated many times in human, bovine and porcine elastin molecules, but not present in the chicken elastin molecule [2, 7]. The hydrophobic repeating peptides of elastin VGVAPG are able to generate directed cell movements of human monocytes, bovine ligamentum nuchae fibroblasts [8], aortic endothelial cells [9] and murine lung carcinoma cells [10]. The interaction of the elastin peptides with these cells is thought to be mediated by a high-affinity cell receptor for VGVAPG sequences, which is related to membrane-bound protein kinase C (PKC) activity [10, 11]. We have previously reported that synthetic elastin peptide VPGVG stimulates the proliferation of chick vascular smooth muscle cells and downregulates elastin expression [12]. In this study, we evaluated the effects of two synthetic elastin peptides VPGVG and VGVAPG, both of which are present in human elastin molecules, on the biosynthetic phenotype of extracellular matrix proteins and on the proliferation of human dermal fibroblasts. The following chemicals were purchased from Seikagaku Corporation, Tokyo: the inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) [13]; the inhibitor for cAMPand cGMP-dependent protein kinase, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004) [14]; the calcium-calmodulin binding inhibitor, N-(6-amino-hexyl)-5-chloro1naphthalenesulfonamide (W-7) [15], and the inhibitor for cAMP-dependent protein kinase, (N-[2-(P-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) [16]. Heparin and 2-O-tetradecanoylphorbol 13-acetate (TPA) were purchased from Sigma. The peptides, VPGVG and VGVAPG were synthesized by a solid-phase method. Polymers of VPGVG and VGVAPG [(VPGVG)n, and (VGVAPG)n, respectively] were synthesized as previously deShingo Tajima · Hiroshi Wachi · Yuko Uemura · Kouji Okamoto

Stimulation of cell proliferation and autoregulation of elastin expression by elastin peptide VPGVG in cultured chick vascular smooth muscle cells

Synthetic elastin peptides, VPGVG or its polymer (VPGVG) n , enhanced the proliferation of smooth muscle cells 1.5‐fold during 48 h treatment at the concetrations over 10−6 M or 1.0 μg/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one‐third and one‐half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin‐specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Elastin synthesis is inhibited by angiotensin II but not by platelet-derived growth factor in arterial smooth muscle cells

Effects of two major potent vasoconstrictors, angiotensin II and platelet-derived growth factor, on elastin expression in cultured chick embryonic arterial smooth muscle cells were studied. Platelet-derived growth factor exhibited no effect on elastin synthesis nor its mRNA level but stimulated (1.5-fold) cell proliferation slightly. Angiotensin II inhibited elastin synthesis dose- and time-dependent manner with a maximum suppression of sixty percent of control at a concentration of 10 microM for 18 h treatment. The suppression was accompanied with a comparable decrease in elastin mRNA level. The inhibition was blocked by addition of Sar1,Ala8-angiotensin II and 8-bromo-cGMP. It showed no effect on cell proliferation. Angiotensin II appears to inhibit elastin synthesis through the interaction with its receptor and the modulation of intracellular Ca2+ level. Thus angiotensin II, not platelet-derived growth factor, can exert a profound effect on the extracellular matrix composition in arterial walls, leading to an arterial change in hypertension or atherosclerosis.

Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Quinazolinobenzodiazepine Derivatives, Novobenzomalvins A-C: Fibronectin Expression Regulators from Aspergillus novofumigatus.

Three new quinazolinobenzodiazepine derivatives, novobenzomalvins A (1), B (2), and C (3), have been isolated as fibronectin expression regulators from Aspergillus novofumigatus CBS117520. The structures of 1 to 3 were established by spectroscopic and physicochemical analysis, and chemical investigation including the total synthesis of 1. Treatment with novo-benzomalvins A (1), B (2), C (3), and N-methylnovobenzomalvin A (5) increased the expression of fibronectin in normal human neonatal dermal fibroblast cells.

Enhanced tropoelastin-degrading activity during cell passages in cultured smooth muscle cells

Tropoelastin expression in vascular smooth muscle cells during serial cell passages from the primary to the tertiary culture was studied. The level of tropoelastin was found to be greatly reduced as the number of cell passages increased. The translational activity and level of elastin mRNA were essentially unchanged throughout the cell passages. The reduction in tropoelastin expression was not due to the repetitive trypsin treatment nor to the prolyl hydroxylation level of the newly-synthesized elastin. A comparable decline in tropoelastin expression was also found with increasing cell division in the primary cultures plated at different cell densities. A pulse-chase experiment revealed that the newly-synthesized elastin in the tertiary culture degraded more rapidly than that in the primary culture. The culture medium harvested from the tertiary culture exhibited a higher tropoelastin-degrading activity than that from the primary culture in the test-tube. The degrading activity of the tertiary culture was inhibited by the addition of 1 mM ethylenediaminetetraacetic acid or 1 mM phenylmethylsulfonyl fluoride, but not by 1 mM N-ethylmaleimide. These results suggest that the reduction in tropoelastin expression during the cell passages from the primary to the tertiary culture is due to the enhanced tropoelastin-degrading activity of the tertiary culture. The transition to tropoelastin-degrading phenotype during cell passages may explain the biological mechanisms of smooth muscle cell migration from the media to the intima observed in the pathological conditions.