Matrix metalloproteinases (MMPs) are hypothesized to play a key role in the processes of endothelial cell migration and matrix remodeling during angiogenesis. We utilized an in vitro model of microvascular endothelial cell angiogenesis, cells cultured within a collagen matrix, to investigate the MMP profile of endothelial cells undergoing angiogenesis. We demonstrated by gelatin zymography that monolayer cultures (two-dimensional) of endothelial cells constitutively expressed low levels of latent MMP-2, but that culture in a three-dimensional collagen matrix increased the total amount of MMP-2 mRNA and protein. Furthermore, 51% of total MMP-2 protein was activated in the three-dimensional culture lysates, compared with 3.5% in two-dimensional culture. The mRNA and protein of MT1-MMP, the putative activator of MMP-2, were up-regulated in endothelial cells cultured in three-dimensional as compared with two-dimensional culture. Treatment of cultures with MMP inhibitors blocked activation of MMP-2 and inhibited formation of endothelial cell networks within the collagen gel. Induction of MT1-MMP and MMP-2 appeared to be specific to collagen, inasmuch as culture of the endothelial cells on top of, or within, a Matrigel(R) matrix neither increased total MMP-2 nor increased activation of MMP-2. These results suggest that MT1-MMP activation of MMP-2 occurs in endothelial cells undergoing angiogenesis, that this activation has a functional role in endothelial cell organization, and that specific matrix interactions may be critical for the increased expression of MT1-MMP and MMP-2.

Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 ± 0.7- and 1.5 ± 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 ± 0.08 in GM-6001-treated muscles compared with 1.69 ± 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 ± 0.27% in GM-6001-treated muscles compared with 3.72 ± 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 ± 9.3 nuclei in control and 63.5 ± 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.

Matrix metalloproteinase activity is instrumental in processes of cellular invasion. The interstitial invasion of endothelial cells during angiogenesis is accompanied by up-regulation of several matrix metalloproteinases, including membrane type 1 matrix metalloproteinase (MT1-MMP). In this study, we show that endothelial cells stimulated to undergo angiogenesis by a three-dimensional extracellular matrix environment increase production of the transcription factor Egr-1. Increased binding of Egr-1 to the MT1-MMP promoter correlates with enhanced transcriptional activity, whereas mutations in the Egr-1 binding site abrogate the increased transcription of MT1-MMP in the stimulated cells. These data identify Egr-1-mediated transcription of MT1-MMP as a mechanism by which endothelial cells can initiate an invasive phenotype in response to an alteration in extracellular matrix environment, thus functionally associating MT1-MMP with a growing number of proteins known to be up-regulated by Egr-1 in response to tissue injury or mechanical stress.

We examined the morphological parameters of arteriolar endothelial and smooth muscle cell dimensions and gap junctional surface areas to obtain an indication of the coupling capacity of each cell type. Silver nitrate staining was utilized to define cell borders of endothelial and smooth muscle cells in arterioles of several vascular beds from two species. From video images of silver-stained arterioles, the mean endothelial cell length of hamster cheek pouch arterioles (diameter 20 to 110 microns) was found to be 141 +/- 2 microns. Mean endothelial cell width was 7 +/- 0.2 microns in the same arterioles. Mean smooth muscle cell length in hamster cheek pouch arterioles of diameter 80 to 150 microns was 66 +/- 3 microns, with an average cell width of 8 +/- 0.2 microns. Dimensions of both endothelial and smooth muscle cells varied moderately with arteriole size and tissue type, but no general trends were seen. Based on the measured dimensions and the specific orientation of cell types within the arteriole, it was calculated that in hamster cheek pouch arterioles (60 microns diameter), 6 or 7 endothelial cell lengths would constitute a 1-mm segment of vessel, whereas approximately 140 smooth muscle cell widths would be required to span the same length. Estimates of connexin43 gap junctional plaque surface areas in each cell type suggest that endothelial cell junctional surface area is approximately eight times that of smooth muscle cells. Thus, combined measurement of cell dimensions and orientation with estimates of junctional plaque density leads to the conclusion that the endothelial cell layer forms a more permissive pathway for longitudinal conduction of signals through the blood vessel.

Capillary growth in skeletal muscle occurs via the dissimilar processes of abluminal sprouting or longitudinal splitting, which can be initiated by muscle stretch and elevated shear stress, respectively. The distinct morphological hallmarks of these types of capillary growth suggest that discrete sets of angiogenic mediators play a role in each situation. Because proteolysis and proliferation are two key steps associated with capillary growth, we tested whether differences in the regulation of matrix metalloproteinases (MMPs) or VEGF may be associated with the two types of capillary growth. We found significant increases in MMP-2 total protein and percent activation, and membrane type-1 MMP mRNA levels, compared with controls after muscle stretch but not after shear stress stimulation. In contrast, VEGF protein and endothelial cell proliferation increased after either angiogenic stimulus. We observed that MMP-2 regulation occurs independent of VEGF signaling, because VEGF did not induce MMP-2 production or activation in isolated endothelial cells. Our data suggest that the involvement of MMPs in capillary growth is dependent on the nature of the angiogenic stimulus.

The process of new blood vessel growth, angiogenesis, involves orchestrated alterations in endothelial cell interactions with adjacent cells and with components of the underlying basement membrane matrix. The activity of matrix metalloproteinases (MMPs), proteases that can cleave basement membrane and interstitial matrix molecules, has been shown to be necessary for angiogenesis as it occurs in several different in vivo and in vitro models. This review discusses the potential roles of two particular MMPs, MMP-2 and MT1-MMP, in angiogenesis, with emphasis on current understanding of how endothelial cell-extracellular matrix interactions may regulate the production of these MMPs via matrix-induced signaling leading to transcriptional activation and subsequent formation of active multiprotease complexes on the cell surface.