Membrane-Type Matrix Metalloproteinases in Human Dermal Microvascular Endothelial Cells: Expression and Morphogenetic Correlation Vincent T. Chan, Dan.

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Membrane-Type Matrix Metalloproteinases in Human Dermal Microvascular Endothelial Cells: Expression and Morphogenetic Correlation Vincent T. Chan, Dan Ning Zhang, Usha Nagaravapu, Kevin Hultquist, Luz I. Romero, G. Scott Herron Journal of Investigative Dermatology Volume 111, Issue 6, Pages 1153-1159 (December 1998) DOI: 10.1046/j.1523-1747.1998.00416.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Angiogenic agents increase expression of MT-MMP-1. Northern blot analysis of 5 μg HDMEC total RNA hybridized with MT-MMP-1 cDNA probe (A). The same blot was stripped and rehybridized with glyceraldehyde 3 dehydrogenase probe (B).Lane 1, untreated HDMEC;lane 2, 16 h PMA-treated HDMEC;lane 3, 20 U IL-1b;lane 4, 10 ng TNF-α;lane 5, 500 ng VEGF 121;lane 6, 20 ng bFGF; andlane 7, 5 μg untreated HT1080 RNA. Transcript sizes are indicated byarrows on the left-hand size. (C) Images from (A) and (B) were densitometrically scanned, values were normalized to glyceraldehyde 3 dehydrogenase and expressed as a ratio of the untreated HDMEC value (lane 1). Numbers above each bar represent fold increase MT-MMP signalversus lane. Journal of Investigative Dermatology 1998 111, 1153-1159DOI: (10.1046/j.1523-1747.1998.00416.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Evidence for multiple processed forms of HDMEC MT-MMP-1. Immunoblots of cellular lysates (5 μg, unless otherwise stated) using four different MT-MMP-1 antibodies: (A) monoclonal MTAb-1 (clone114–1F2); (B) monclonal MTAb-3 (clone 114–6G6); (C) polyclonal MTAb-2 (directed against the same MT-MMP-1 epitope); (D) polyclonal MTAb-4 (directed against the “hinge region” of MT-MMP-1). Blotting conditions and antibody dilutions were performed according toMaterials and Methods. (A) Ulex(–) untreated dermal cells (lane 1), untreated PECAM-affinity purified HDMEC (lane 2), 16 h PMA-treated HDMEC (lane 3), and untreated HT1080 lysate (lane 4). (B) One microgram untreated HDMEC lysate (lane 1), 3 μg untreated HDMEC lysate (lane 2), 5 μg untreated HDMEC lysate (lane 3), and 5 μg untreated HT1080 lysate (lane 4). (C) Untreated HT1080 (lane 1), untreated HDMEC (lane 2), and 16 h PMA-treated HDMEC (lane 3). (D) One microgram untreated HDMEC lysate (lane 1), 3 μg untreated HDMEC lysate (lane 2), 5 μg untreated HDMEC lysate (lane 3), 10 μg untreated HDMEC lysate (lane 4), 10 μg untreated HT1080 lysate (lane 5), and 10 μg of untreated Ulex(–) cell lysate (lane 6). “Apparent” molecular weight standards in kilodaltons are indicated byarrows on the left-hand side. Major immunoreactive species are indicated byarrows on the right-hand side labeled as 63/65 (63–65 kDa), 57/60 (57–60 kDa), 50/53 (50–53 kDa), and 43/44 (43–44 kDa). Journal of Investigative Dermatology 1998 111, 1153-1159DOI: (10.1046/j.1523-1747.1998.00416.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 HDMEC MT-MMP-1 converts proMMP2 to its active form. Zymographic analysis of gelatinase activation by HDMEC membrane extracts. Seventy-two hours incubation of gelatinases (proMMP-2 at 72 kDa and proMMP-9 at 92 kDa) with detergent extraction buffer (1.5% Triton X114 in TBS) alone (lane 1), or with untreated HDMEC membrane extract (lane 2), 0.75% paraformaldehyde-treated HDMEC extract (lane 3), monoclonal antibody 114–1F2 treated HDMEC extract (lane 4), or combination antibody 114–1F2 plus 0.75% paraformaldehyde HDMEC extract (lane 5) as described inMaterials and Methods. Appearance of activation products at 67 kDa and 84 kDa indicated on the right-hand side correspond to activated forms of MMP-2 and MMP-9, respectively. Separate panel shows densitometric analysis of zymogram,lanes 2–5. Whereas other species remain essentially unchanged, specific decrease of the 67 kDa MMP-2 activation product is evident inlane 5 and represents an over 2-fold reductionversus controllane 2. Journal of Investigative Dermatology 1998 111, 1153-1159DOI: (10.1046/j.1523-1747.1998.00416.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Antibodies to MT-MMP-1 blockin vitro angiogenesis. The time course of three-dimensional collagen tubule assay. Monolayer HDMEC were exposed to three-dimensional collagen gel at “time 0” after 12 h pretreatment with a nonendothelial cell surface monoclonal antibody LAD-1 (a–c, “control”) or with monoclonal antibody MTAb-1 (114–1F2) pretreatment (d–f, “anti-MT-MMP-1”). HDMEC normally respond to three-dimensional collagen by rapid movement to form “tubule-like” structuresin vitro. Journal of Investigative Dermatology 1998 111, 1153-1159DOI: (10.1046/j.1523-1747.1998.00416.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions