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Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and
L A Cornelius1, L C Nehring, J D Roby
1Department of Medicine, Jewish Hospital, Washington University, St. Louis, Missouri 63110, USA.
Abstract:
Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.
Insights
Microvascular endothelial cells (MECs) uniquely regulate matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). This specific expression profile is crucial for cell migration, wound healing, and angiogenesis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Matrix metalloproteinases (MMPs) degrade extracellular matrix, facilitating cell migration.
- Tissue inhibitors of metalloproteinases (TIMPs) regulate MMP activity.
- Endothelial cell MMP expression is critical for neovascularization.
Purpose of the Study:
- Characterize metalloenzyme production by microvascular endothelial cells (MECs).
- Investigate the modulation of MMP and TIMP expression by phorbol esters (PMA) and cytokines (TNF-alpha, FGF, IFN-gamma).
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA)
- Immunoprecipitation
- Northern hybridization
- Transfection assays
- In situ hybridization
Main Results:
- MECs exhibit low constitutive MMP expression.
- PMA stimulates MMPs and TIMP-1, down-regulates TIMP-2.
- TNF-alpha induces cell-specific collagenase upregulation and inhibits PMA-induced TIMP-1.
- IFN-gamma upregulates TIMP-1 and inhibits collagenase induction.
- Regulation occurs at pretranslational and transcriptional levels.
- Collagenase mRNA is expressed by migrating MECs.
Conclusions:
- MECs possess a unique MMP/TIMP expression and regulation profile compared to other cell types.
- This profile is vital for wound healing and angiogenesis, especially during early migration phases.