Abstract
We have previously reported that vitamin A inhibits the growth of capillary endothelial cells (EC) (Braunhut, S.J., and Palomares, M. (1991) Microvasc. Res. 41, 47-62). In this study, we have analyzed the effect of retinoids on the production and activity of enzymes involved in the proteolytic degradation of extracellular matrix (ECM) by EC. Substrate gel electrophoresis (zymography) revealed several major matrix metalloproteinases (MMP), of approximately 98-96, 72-68, and 46-45 kDa, whose activities were altered in their amounts in the conditioned media (CM) of EC following retinol or retinoic acid treatment when compared to amounts detected in CM of control cells. All of these gelatinases were inactivated by 1,10-phenanthroline, indicating that they were MMPs. MMP inhibitors (MMPI) were also present in these CM and were separated by gel filtration. Four distinct peaks of MMPIs were detected in the CM of EC. Chromatographic profiles indicated that an approximately 27-kDa MMPI was specifically increased in the CM of retinol-treated cells, whereas a 22-18.5-kDa MMPI was increased in CM derived from retinoic acid-treated cells. The MMPI synthesized by retinol-treated EC was immunologically related to tissue inhibitor of metalloproteinases, type 1 (TIMP-1), and the MMPI produced by retinoic acid-treated cells was related to TIMP-2, as indicated by biosynthetic labeling and immunoprecipitation studies as well as Western blot analysis. Therefore, retinol and retinoic acid treatment of EC differentially affects the types and activity of MMPs and MMPIs produced by the cells, distinct changes which are both correlated with growth inhibition.
Highlights
We have previously reported that vitaminA inhibits chronically exposed to circulating vitamin A, little is known the growthof capillary endotheliaclells (EC)
The MMPI produced by retinoic acid-treated cells was The activity of matrix metalloproteinases (MMP), a multirelated toTIMP-2,as indicated by biosynthetic labeling gene family of metal-dependent enzymes, is the rate-limiting andimmunoprecipitationstudies as well as Western step in ECM degradation
The endogenous inhibitors of the MMPs are plexed to a retinol binding protein synthesized there (Goodman known as tissue inhibitor of metalloproteinases (TIMP)
Summary
The Morphology of E C Is Differentially Altered following Exposure to Retinol and Retinoic Acid (RA)-In the presentstudy, EC treated with retinol or F U for 6 days were 100% growthinhibited by day 6 of culture, as previously reported Growth medium alone contained pg/ml total protein This amountw, e found that ECs, regardlesosf treatment group, secreted approximately the same amount of protein into the FIG2. Densitometry was a variety of proteins not found in medium alone (lane 1), indi- performed on the upper two prominent bands of activity and cating that these protein species were produced by the ECs. demonstrated that retinol-treated EC had 1.4 times and RA-. The samplesshown Computer densitometry revealed that theCM derived from an in Fig. 3 represents theCM protein from 3.56 x lo4cells of each equivalent numberof retinol-treated cells contained 2.46 times treatment group and an equivalent amounotf protein derived more (lane 3 ) and RA-treated EC CM had significantly less from the sampleof concentrated CM alone processed in paral- (0.74 times) (lane4 ) of this proteolytic activity, the M, = 72,000 lel. Proteolytic activities migratedwith a molecular weight consist- An additional, single band of proteolytic activitywas obent with the reported molecular weight for MMP-9 (Type V served in the CMof control EC (lane 2 ) a t approximately 46
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