Tumor cells become malignant, in part, because of their activation of matrix metalloproteinases (MMPs) and inactivation of tissue inhibitor of metalloproteinases (TIMPs). Myocardial tumors are rarely malignant. This raises the possibility that the MMPs and TIMPs are differentially regulated in the heart compared to other tissues. Therefore, we hypothesized that a tissue specific tumor suppressor exists in the heart. To test this hypothesis we prepared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic (ICM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-stage explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhibitory activity of chromatographic peaks was determined using fluorescein-conjugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIMP-4 in cardiac fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast cells migration and collagen gel invasion. To test whether TIMP-4 induces apoptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measured and DNA laddering was determined. The results suggested that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells. Cardiospecific TIMP-4 plays a significant role in regulating the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (remodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to malignancy.

2001.-To test the hypothesis that homocysteine induces constrictive vascular remodeling by inactivating peroxisome proliferator-activated receptor (PPAR), aortic endothelial cells (ECs) and smooth muscle cells (SMCs) were isolated. Collagen gels were prepared, and ECs or SMCs (10 5 ) or SMCs ϩ ECs (10 4 ) were incorporated into the gels. To characterize PPAR, agonists of PPAR-␣ [ciprofibrate (CF)] and PPAR-␥ [15-deoxy-12,14-prostaglandin J 2 (PGJ2)] were used. To determine the role of disintegrin metalloproteinase (DMP), cardiac inhibitor of metalloproteinase (CIMP) was used in collagen gels. Gel diameter at 0 h was 14.1 Ϯ 0.2 mm and was unchanged up to 24 h as measured by a digital micrometer. SMCs reduce gel diameter to 10.5 Ϯ 0.4 mm at 24 h. Addition of homocysteine to SMCs reduces further the gel diameter to 8.0 Ϯ 0.2 mm, suggesting that SMCs induce contraction and that the contraction is further enhanced by homocysteine. Addition of ECs and SMCs reduces gel diameter to 12.0 Ϯ 0.3 mm, suggesting that ECs play a role in collagen contraction. Only PGJ 2, not CF, inhibits SMC contraction. However, both PGJ 2 and CF inhibit contraction of ECs and SMCs ϩ ECs. Addition of anti-DMP blocks SMC-as well as homocysteine-mediated contraction. However, CIMP inhibits only homocysteine-mediated contraction. The results suggest that homocysteine may enhance vascular constrictive remodeling by inactivating PPAR-␣ and -␥ in ECs and PPAR-␥ in SMCs. aorta; arteriosclerosis; hypertension; peroxisome proliferator-activated receptor; fibrate; prostaglandin; endothelial cell; smooth muscle cell ARTERIAL WALL REMODELING is one of the most important factors regulating lumen diameter after acute and/or chronic vascular injury (7,28,29,44). Smooth muscle cells (SMCs) remodel the existing and new extracellular matrix (ECM). In response to ECM degradation, SMCs alter phenotype (43). The consequences of remodeling may lead to alterations in arterial wall geometry and lumen diameter (7,28,29,44). Although the extracellular environment strongly influences cell behavior, it is unclear whether the changes in matrix composition affect connective tissue shrinkage. Hyperhomocysteinemia is associated with hypertension (39) and increases vascular intimal-medial thickness (26,34). Homocysteine causes arteriosclerosis (19,36,40), endothelial cell desquamation (38), thromboresistance (22), SMC proliferation (41, 45), collagen synthesis (23, 45), oxidation of low-density lipoprotein (12), increased monocyte adhesion to the vessel wall (20), platelet aggregation (6), coagulation (34), blood rheology (8, 25), and activation of plasminogen and metalloproteinase (18, 47), the two neutral proteinases associated with remodeling. Previous studies from our laboratory have identified a redox-sensitive homocysteine receptor in SMC. This receptor regulates collagen expression (45). Primarily, there are two nuclear transcription factor (NF) receptors that control the redox state of the cell. NF-B is induced by homocysteine (3, 49). Peroxisome proliferator-activat...

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.

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Vibrational circular dichroism spectra have been measured in [2HH,]DMS0 solutions for simple carbohydrates in the 1650-800 cm-' region. The links between the observed spectral features and configurational and conformational aspects of carbohydrates were investigated. Some useful correlations and difficulties involved in deciphering the spectral content are pointed out. Vibrational circular dichroism (v.c.d.) is a measure of differential absorption of left versus right circularly polarized i.r. radiation due to the vibrational transitions of chiral molecules. The unique nature of v.c.d. spectroscopy results from the combination of a large number of vibrational transitions with the stereochemical sensitivity inherent in circular dichroism (c.d.) The conventional c.d. studies are carried out in the visible spectral region, probing the electronic transitions, and such accessible electronic transitions are usually very few in number. For simple carbohydrates, in particular, there are no electronic transitions in the visible spectral region, which required a major portion of optical activity studies on simple carbohydrates to be limited to studying the optical rotation.' Thus v.c.d. spectroscopy offers two advantages for simple carbohydrates. There are 3n -6 fundamental vibrational transitions where n is the number of atoms in a molecule, and every one of these transitions can, in principle, exhibit c.d.; this provides a very large base of accessible spectral transitions. Secondly, since different vibrations encompass different portions of the molecule, c.d. features associated with different vibrational bands provide three-dimensional structural information in different portions of the molecule. Thus a complete v.c.d. spectrum contains the necessary information about the three-dimensional structure of the entire molecule in the solution phase. In practice, though, the determination of the complete molecular structure from v.c.d. spectra is rather ambitious, with the current state of knowledge, owing to (i) the complicated nature of molecular vibrations, (ii) the overlapping of vibrational bands and (ili) the small v.c.d. signals that are beyond instrumental sensitivity.In this paper we present some of the v.c.d. spectral studies carried out in our laboratory in the last five years in the 1650-800 cm-I region. V.c.d. spectra of lactones, sorbose and some additional simple carbohydrates were also investigated in our laboratory, and they will be reported separately. A preliminary report from our laboratory2 and two other v.c.d. paper^^^^ in the C-H stretching region comprise the previously published v.c.d. studies for carbohydrates. Experimental The v.c.d. spectrometer used for the present studies is based on a commerical Fouriertransform i.r. spectrometer (Nicolet 6000C). The instrumental details for these v.c.d. measurements are given el~ewhere.~ The raw v.c.d. features are usually dominated by the artifacts resulting from the birefingence in the optical path, which makes it difficult to determine the zero line. It is...