Membrane-anchored proteases in endothelial cell biology

Abstract

Purpose of review

The endothelial cell plasma membrane is a metabolically active, dynamic and fluid microenvironment where pericellular proteolysis plays a critical role. Membrane-anchored proteases may be expressed by endothelial cells, as well as mural cells and leukocytes with distribution both inside and outside of the vascular system. Here we will review the recent advances in our understanding of the direct and indirect roles of membrane-anchored proteases in vascular biology and the possible conservation of their extravascular functions in endothelial cell biology.

Recent findings

Membrane-anchored proteases belonging to the serine or metalloprotease families contain amino- or carboxy-terminal domains which serve to tether their extracellular protease domains directly at the plasma membrane. This architecture enables protease function and substrate repertoire to be regulated through dynamic localization in distinct areas of the cell membrane. These proteases are proving to be key components of the cell machinery for regulating vascular permeability, generation of vasoactive peptides, receptor tyrosine kinase transactivation, extracellular matrix proteolysis and angiogenesis.

Summary

A complex picture is emerging of the interdependence between membrane-anchored protease localization and function that may provide a mechanism for precise coordination of extracellular signals and intracellular responses through communication with the cytoskeleton and with cellular signaling molecules.

Introduction

Endothelial cells regulate communication between the blood and the subendothelial tissues critical for a wide range of important physiological processes including hemostasis, the transport of solutes and macromolecules, regulation of vasomotor tone, angiogenesis, and the trafficking of leukocytes for innate and adaptive immunity [1]. The endothelial cell plasma membrane is a metabolically active, dynamic and fluid microenvironment where pericellular proteolysis plays a critical role. For many years, investigative studies have largely focused on the proteolytic enzymes that mediate pro-coagulant and anti-coagulant activities, given the critical importance of endothelial cells in maintaining hemostatic balance. The activities of secreted, pericellular coagulation proteases (e.g. thrombin, Factor Xa), the plasminogen system (e.g. tPA, uPA and plasmin) and the matrix metalloproteinases (MMPs) are well studied in this regard. Recently there has been an increase in the identification of plasma membrane-tethered proteases, such as the family of membrane-anchored serine proteases, the ADAM family, and the membrane-type MMPs (MT-MMPs), whose direct and indirect roles in vascular functions, blood vessel growth, development and structure are becoming evident through studies of murine deficiencies and dysregulation in human diseases (Table 1). The membrane-anchored proteases are widely distributed both inside and outside of the vascular system and may be expressed by endothelial cells, mural cells, and/or leukocytes. Membrane anchorage enables these proteases to initiate pericellular proteolysis in the cell microenvironment, and to interact with membrane proteins on the same cells or on nearby cells. The specific functions of many of these proteases in vascular biology, and in some cases, even their expression by endothelial and mural cells, are poorly understood. In this review, we will focus on the current knowledge of membrane-anchored proteases in vascular biology with an emphasis on recent findings, and the possible conservation of their extravascular functions in endothelial cell biology.

Table I.

Membrane-anchored proteases associated with endothelial and vascular biology and their functions.

Protease	Common Aliases	Gene Symbol	Membrane Anchor	In Vivo Vascular and Extravascular Functions	Ref.
Serine Proteases
Membrane-anchored Serine Proteases
Prostasin	PRSS8, CAP1	PRSS8	GPI	Epidermal deficiency causes defective barrier function in mice. Tissue specific deletion in mice shows reduced fluid clearance in lung and colonic epithelium due to defects in ENaC activation. Liver specific knock-out mice develop insulin resistance associated with associated with reduced hepatic TLR-4 shedding. Implicated in blood pressure regulation in humans.	[2–8]
Matriptase	MT-SP1, CAP3, ST14, PRSS14, epithin	ST14	Type II	Null and hypomorphic mice show defective global epithelial barrier function due to increased paracellular permeability. Activates the c-Met ligand pro-HGF/SF when over expressed in keratinocytes in mice.	[9–11]
Matriptase-2	TMPRSS6	TMPRSS6	Type II	Essential for iron homeostasis by cleavage of hemajuvelin, prevents iron-refractory iron deficiency anemia in humans. Over-expression in human vascular endothelial cells inhibits tumor induced angiogenesis in a murine xenograft model.	[12,13]
Corin	LRP4, ATC2, TMPRSS10	CORIN	Type II	Important regulator of blood pressure and volume via the activation of natriuretic peptides in cardiomyocytes. Promotes uterine spiral artery remodeling during pregnancy.	[14–16]
Proprotein convertases
Furin	Paired basic amino acid cleaving enzyme, FUR, PACE, SPC1, PCSK3	FURIN	Type I	Null mice are embryonic lethal due to a failure of ventral closure and large blood vessel development. Endothelial cell specific knockout mice die due to cardiovascular dysfunction.	[17,18]
Prolyl oligopeptidases (POP)
DPPIV	Dipeptidyl peptidase 4, CD26	DPP4	Type II	Regulates vascular endothelial cell function. Inactivates stromal cell-derived factor-1 (SDF-1), a key regulator of vascular stem cell homing. Inhibition lowers blood pressure, decreases cardiovascular disease and improves outcomes after myocardial infarction in mice.	[19–22]
Metalloproteases
Membrane-type
MT1-MMP	MMP14, MT-MMP1	MMP14	Type I	Null mice show reduced vascular invasion of cartilage and defective corneal angiogenesis in response to basic fibroblast growth factor.	[23–25]
MT4-MMP	MMP17, MT-MMP4	MMP17	GPI	Deficiency predisposes mice to aortic aneurysm due to dysfunctional vascular smooth muscle cells. Loss of expression in patients with hereditary thoracic aortic aneurysms increases susceptibility to aneurysm rupture.	[26**]
ADAM Proteases
ADAM8	CD156, cell surface antigen MS2, human leukocyte differentiation antigen	ADAM8	Type I	Null mice show increased branching and vessel density in pathological retinal neovascularization assays.	[27–29]
ADAM9	meltrin gamma, cellular disintegrin- related protein	ADAM9	Type I	Murine deficiency inhibits pathological retinal neovascularization in oxygen-induced retinopathy and laser-induced choroidal models.	[27,30]
ADAM10	CD156c, kuzbanian protein homolog, mammalian disintegrin- metalloprotease	ADAM10	Type I	Murine deficiency is embryonic lethal due to defective blood vessel development. Endothelial cell specific deficiency, or inhibition of ADAM10, causes increased pathological retinal neovascularization following oxygen-induced retinopathy.	[27,31, 32*]
ADAM12	Meltrin alpha, metalloprotease- disintegrin 12 transmembrane	ADAM12	Type I	Knockdown of ADAM12 in mice inhibits recovery from experimental peripheral artery disease. Inhibition of ADAM12 in mice reduces hypoxia-induced impairment of neural vascular barrier function.	[2,27,33,34**]
ADAM15	Metargidin, a disintegrin and metalloproteinase domain 15	ADAM15	Type I	Null mice or mice carrying an active site mutation, show decreased pathological retinal neovascularization following oxygen-induced retinopathy. Null mice show a reduction in the development of atherosclerotic lesions caused by apolipoprotein E-deficiency. Mediates endothelial hyperpermeability during acute lung injury induced by lipopolysaccharide in mice.	[27,35– 37]
ADAM17	TNF-alpha converting enzyme (TACE), ADAM metallopeptidase domain 18	ADAM17	Type 1	Murine deficiency or mice lacking the ADAM17 Zn binding site have defective blood vessel formation causing death. Endothelial cell specific deficiency or ADAM17 inhibition causes reduced pathological retinal neovascularization. Inhibition in mice reduces hypoxia-induced impairment of neural vascular barrier function.	[2,27,32,34,38]
ADAM33	disintegrin and reprolysin metalloproteinase family protein	ADAM33	Type I	Susceptibility gene for asthma and chronic obstructive pulmonary disorder. Soluble form is found in asthmatic bronchiolar lavage fluid and promotes human endothelial cell differentiation and tube formation in vitro, and neovascularization in the chorioallantoic membrane assay. Also implicated in the pathogenesis of atherosclerosis.	[27,39– 41]
Angiotensin- converting enzymes
ACE	angiotensin- converting enzyme, ACE1, CD143, carboxycathepsin, dipeptidyl carboxypeptidase 1	ACE	Type I	Catalyzes the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor causing increased blood pressure. ACE inhibitors are the primary treatment for hypertension. Over-expression is correlated with cardiovascular diseases.	[42,43]
ACE2	angiotensin- converting enzyme 2, ACEH, ACE- related carboxypeptidase	ACE2	Type I	Acts as a counter-regulatory mechanism to ACE1 by cleaving angiotensin II to angiotensin products with vasodilator and vasoprotective properties.	[44,45]

Abbreviations: ADAM, a disintegrin and metalloproteinase; ENaC, epithelial sodium channel; MMP, matrix metalloproteinases; MT-MMP, membrane tethered-matrix metalloprotease; pro-HGF/SF, pro-hepatocyte growth factor/scatter factor; PRSS, protease serine S1; PSCK, proprotein convertase subtilisin/kexin type; TLR, toll-like receptor; TMPRSS, transmembrane protease serine S1.

Membrane anchoring domains

The membrane-anchored proteases associated with the vasculature and discussed here all belong to the serine protease or metalloprotease families (Figure 1). The unique feature of these membrane-anchored protease families that distinguishes them from secreted soluble proteases such as MMPs and blood coagulation proteases, is that they contain domains which tether them directly to the cell surface, with their catalytic domains exposed to the extracellular environment. The manner in which they are linked to the cell surface may be through Type I or Type II single pass transmembrane domains, or via glycophosphatidylinositol (GPI)-anchors [47,49–52] (Table 1). All of these membrane-anchored proteases are synthesized with an N-terminal signal sequence which directs their trafficking to the cell surface. Type I membrane-anchored proteases are then anchored via a C-terminal hydrophobic region which spans the plasma membrane. Similarly, GPI anchored proteases possess a C-terminal hydrophobic region, which is modified by the addition of a GPI lipid moiety that anchors the protease to cholesterol rich regions in the plasma membrane. In Type II membrane-anchored proteases the N-terminal signal sequence is not removed after trafficking to the cell surface, and serves to anchor the N-terminus to the cell membrane, with the C-terminal catalytic domain localized to the extracellular space. In comparison to secreted proteases this surface localized context provides a higher degree of specificity in the substrates these proteases will encounter, with a requirement that they be either found co-localized on the cell surface or in the nearby pericellular environment. The type of membrane anchor can serve to target these proteases to specific membrane localizations (eg. apical, basolateral, cell junctions and cell protrusions) and microdomains (eg. lipid rafts), and their cytoplasmic extensions are frequently associated with membrane trafficking and/or signal transduction. In some situations, and frequently in response to inflammatory stimuli, the extracellular domains of membrane-anchored proteases are shed from the cell surface [47,49,53], which may indicate a down-regulation of the proteolytic functions of these enzymes. In addition, other mechanisms for regulation of cell surface protease activities exist, including natural inhibitors such as Kunitz-type inhibitors and serpins for the serine proteases and TIMPs for the metalloproteases, and protease endocytosis and turnover [47,54].

Figure 1. Domain structures of the membrane-anchored serine proteases and metalloproteases associated with endothelial and vascular biology.

The serine-type proteases are comprised of the membrane-anchored serine proteases, the proprotein convertases and the prolyl oligopeptidases. The metalloproteases consist of the membrane-type MMPs (MT-MMPs), the ADAMs (a disintegrin and metalloproteinase) and the angiotensin-converting enzymes (ACE). The catalytic domains, which function to hydrolyze the peptide bonds in protein substrates, are all located extracellularly. The catalytic domains of the membrane-anchored serine proteases are either chymotrypsin-like (S1 family), bacterial subtilisin-like (S8 family) or related to the prolyl oligopeptidases (S9 family). They all share the conserved catalytic triad amino acid residues His, Asp and Ser, albeit in different configurations. There are a total of 17 human type II transmembrane serine proteases, synthesized with a unique stem region adjacent to the plasma membrane, containing diverse protein-protein interaction domains such as scavenger receptor cysteine-rich (SRCR), CUB, SEA and Frizzled domains [46]. There are two human GPI-anchored serine proteases, testisin and prostasin. The proprotein convertases are all Type I transmembrane serine proteases containing a cysteine rich region and conserved P-domains which are essential for folding and activation. The prolyl oligopeptidases are synthesized as type II transmembrane homodimeric glycoproteins. The membrane-anchored metalloproteases differ from the membrane-anchored serine proteases in that they utilize a zinc ion to create a nucleophile for protease catalytic activity. The 6 MT-MMPs expressed in humans are either Type I (MT1-, MT2-, MT3- and MT5-MMP) or GPI-anchored (MT4- and MT6-MMP). All of the MT-MMPs share a similar hinge region and hemopexin domain, and are synthesized with an amino-terminal signal sequence and pro-domain which is cleaved by proprotein convertases during transport from the ER/Golgi to the surface, allowing the MT-MMPs to be expressed on the cell surface as active proteases [47]. The ADAM metalloproteinases share a complex domain structure containing the pro-domain followed by the metalloprotease catalytic domain, a disintegrin-like domain enabling cell-cell interactions via integrins, a cysteine-rich domain and an epidermal growth factor-like domain [48]. The ACE family of metalloproteases includes both ACE and ACE2 transmembrane proteases, which consist of an amino-terminal signal domain followed by a large extracellular domain that consists of either two independent (ACE) or one (ACE2) catalytic domains containing zinc binding motifs. The cytoplasmic extensions vary in length for these membrane-anchored proteases and are involved in membrane trafficking, and localization, with some participating in cell signaling.

Activation of membrane-anchored proteases

Most of the membrane-anchored proteases are synthesized as inactive proteins, which are converted to active protease forms by other proteases, or indirectly activated by cellular stimuli, such as sheer stress, cytokines and growth factors. The serine proteases, with the exception of the prolyl oligopeptidases, require a conformational change which generates an active site following cleavage of a propeptide that remains tethered to the catalytic domain through disulfide linkages [54]. In contrast, the MT-MMPs and the ADAMs are synthesized with a fully functional active site that is shielded by the prodomain until being processed during secretion by proprotein convertases [47,55,56]. Classically ADAMs were thought to be activated by cleavage between the pro- and catalytic domains at a canonical furin cleavage site (RXXR). However recent evidence has identified a second furin cleavage site embedded in the pro-domain that is critical for the activation and catalytic activity of ADAMs 9, 10 and 17 [57]. In addition, ADAM17 requires catalytically inactive members of the rhomboid family of proteases, iRhom1 and iRhom2, to mediate its intracellular transport and maturation, and mutations in iRhom2 may predispose patients to an inherited cancer susceptibility syndrome [58**]. Membrane-anchored serine proteases, such as matriptase, can self-activate or proteolytically activate other precursor serine protease zymogens [54], and further, non-enzymatic stimulation of matriptase activation by prostasin and activation of prostasin by the matriptase zymogen has been reported [59,60], suggesting complex interrelationships between these enzymes.

Ectodomain shedding of cell surface molecules

The ADAM proteases are recognized to play a major role in ectodomain shedding of a number of transmembrane molecules that play pivotal roles in the control of cell adhesion and cell signaling processes [61–65]. ADAM17 is a central regulator of the tumor necrosis factor (TNF) receptor and epidermal growth factor receptor (EGFR) signaling pathways that drive inflammation and injury/repair responses. Although both ADAM17 and ADAM10 have been considered the primary proteases responsible for ectodomain shedding of the Notch receptor during sprouting angiogenesis, a recent study finds that ligand-induced Notch signaling is initiated primarily by ADAM10 [32*]. During this process, ADAM17 appears to promote sprouting angiogenesis by down-regulating the inhibitor of angiogenesis thrombospondin 1 (TSP1) [32*].

Influence of membrane dynamics on substrate accessibility

The actin cytoskeleton is important for controlling membrane-anchored protease localization on the plasma membrane as well as substrate accessibility. Recent studies demonstrate that MT1-MMP and one of its substrates, the heparin-binding adhesion molecule CD44, can co-localize through anchoring to the actin cytoskeleton through ezrin/radixin/moesin (ERM) protein-mediated interactions between their cytoplasmic tails [66]. It remains to be seen whether other membrane proteases and adhesion molecules are linked to the cytoskeleton via intracellular complexes. The cytoskeleton can also mediate substrate accessibility. The transmembrane endothelial adhesion molecule CX₃CL1 exists within confinement regions structured by the cortical actin cytoskeleton, which precludes its association with ADAM10. Disruption of the actin cytoskeleton in response to secreted inflammatory cytokines, leukocyte-endothelial cell interactions or changes in mechanical force exerted by the endothelium can reduce CX₃CL1 sequestration and increase CX₃CL1–ADAM10 interactions, resulting in enhanced shedding of soluble CX₃CL1, a chemoattractant for circulating leukocytes [67**].

Influence of membrane microdomains/lipid rafts on membrane-anchored protease functions

A striking feature of the GPI-anchored proteases is their enrichment in lipid raft membrane microdomains. Testisin is a GPI-anchored serine protease expressed in microvascular endothelial cells [68] that cleaves and activates the seven-transmembrane G-protein-coupled protease-activated receptor, PAR-2, to release its tethered ligand for signal activation [69*]. Interestingly, the activation of PAR-2 by testisin induces internalization of PAR-2 and loss from the cell surface [69*], which has the potential to alter PAR-2 signal activation by soluble proteases that may be present in the extracellular microenvironment. PAR-2 can localize to lipid raft domains; indeed localization of tissue factor (TF) and PAR-2 in lipid rafts is critical for TF:FVIIa to trigger PAR-2-mediated cell signaling [70]. Lipid raft localization has also been found to regulate the cleavage specificity of PAR-1 in endothelial cells, rendering PAR-1 shielded from interaction with coagulation proteases [71]. The importance of lipid raft localization to PAR signaling responses after membrane-anchored protease activation is an interesting area for future investigation.

Vascular permeability barrier

Adjacent endothelial cells are linked by multi-protein intercellular junctional complexes composed of tight junctions, adherens junctions and gap junctions which mediate vascular integrity and control vascular permeability across the paracellular pathway [72]. Vascular permeability is controlled by the oligomerization of the membrane-spanning occludin and claudins, including claudin-5, vascular endothelial cadherin (VE-cadherin) and the endothelial junctional adhesion molecule JAM-A, which interact with cytoplasmic zona occuldens (ZO) proteins in intercellular junctional complexes and link to the actin cytoskeleton [73]. Wide variations in permeability exist across the vascular tree, with capillaries being most permeable and the blood brain barrier being relatively impermeable by comparison. Several ADAMs mediate the shedding of cell-cell adhesion molecules to modulate vascular permeability during excessive inflammation and hypoxia [27,74]. Both ADAM12 and ADAM17 were recently reported to contribute to impaired neural vascular barrier function induced by hypoxia through the degradation of claudin-5 in brain microvascular endothelial cells [34**]. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the loss of barrier function in retinal neural vasculature in vivo under hypoxic conditions. Upon hypoxia induction, it was found that the subcellular localization of ADAM12 and ADAM17 changed rapidly from cytoplasmic to cell membrane-associated, thus enabling a rapid proteolytic response. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the loss of barrier function in retinal neural vasculature in vivo under hypoxic conditions, suggesting their inhibition could offer novel treatment strategies for diseases associated with an impaired neural barriers. ADAMs may also regulate vascular permeability though mechanisms independent of junction molecule shedding, as ADAM15 is reported to induce hyper-permeability of endothelial barriers through Src/ERK1/2 signaling [75,76]. Further, a recent study has identified that the targeting of the 3' UTR of ADAM15 by the microRNA (miR)-147b attenuates ADAM15 expression. Whether miR-147b and/or other microRNAs regulate additional proteases and may be exploited to enhance endothelial barrier protection is an interesting area for further investigation.

Generation of vasoactive peptides

Several membrane-anchored proteases play key roles in cardiovascular remodeling, hypertension and angiogenesis through the cleavage and activation of vasoactive peptides. Corin present on cardiomyocytes converts the atrial natriuretic peptide (ANP) precursor, pro-ANP, to mature ANP, for the regulation of salt-water balance and blood pressure [15]. ANP also protects the endothelial cell barrier against hyperpermeability through its ability to modulate Rho-dependent actin remodeling [77]. Angiotensin converting enzyme (ACE) and ACE2 produce the vasoactive pro-inflammatory peptides Ang-II and its counter-regulatory peptide Ang-(1–7), respectively [78]. This is important because the balance between ACE/Ang II/AT₁ receptor and ACE2/Ang-(1–7)/Mas receptor is critical for vascular homeostasis, especially the regulation of vasoconstriction and proliferation. Overexpression of ACE2 can protect endothelial cell function by promoting endothelial cell migration, and decreasing Ang II induced effects such as pro-inflammatory cytokines and adhesion molecules [79*].

Receptor tyrosine kinase transactivation

Various mechanisms of ligand activation and transactivation of receptor tyrosine kinases depend on membrane anchored proteases. Matriptase is an efficient proteolytic activator of pro-hepatocyte growth factor (HGF), the ligand for the c-Met receptor tyrosine kinase [10,49] that stimulates cell proliferation and promotes activation of pro-angiogenic and vasoprotective pathways in the vasculature [80]. Several of the membrane-anchored proteases, including essentially all of the known ADAMs, shed mature ligands that in turn transactivate receptor tyrosine kinases to promote pro-angiogenic pathways in endothelial cells [27,81–83]. Agonists of multiple G protein-coupled receptors (GPCR) can activate ADAMs to produce mature EGFR ligands that lead to EGFR transactivation; signaling events that may require trafficking and compartmentalization of the GPCR and ADAM providing the temporal and spatial regulation necessary for rapid and specific signal activation [84]. Further, a recent report indicates that heterotrimeric G-proteins may also directly activate membrane tethered proteases in a membrane delimited manner [85*]. MT1-MMP was shown to be activated via a direct binding interaction with activated G-protein βγ subunits, resulting in HB-EGF release and EGFR transactivation [85*]. It remains to be determined whether other membrane-anchored proteases may be activated by the same or different G-proteins in a similarly direct manner.

Membrane regulated and directed ECM proteolysis

During angiogenesis, endothelial cells initiate new blood vessel growth and invasion into the extracellular matrices (ECM). MT1-MMP functions as the major membrane-anchored collagenase, while MT1-MMP, MT2-MMP, and MT3-MMP all have fibrin degrading activities [47,56,86,87]. Shear wall stress stimulates the activation of MT1-MMP by proprotein convertases [88] and its translocation to the plasma membrane at the tips of growing vessels to promote ECM degradation [89]. MT1-MMP and most of the MT-MMPs, with the exception of MT4-MMP, have the demonstrated capability of activating pro-MMP-2 [47,90], which is able to degrade multiple ECM proteins including collagens, fibrin, fibronectin and vitronectin. Studies of MT4-MMP loss-of-function in mice [26**] show that its activity is required for vascular smooth muscle cell maturation and arterial wall function in vivo and its absence results in predisposition to thoracic aortic aneurysms. The phenotype is associated with reduced cleavage of the extracellular matrix protein, osteopontin, important for embryonic development of the aorta. MT1-MMP may also stimulate proMMP-2 activation through paracrine mechanisms. For example, activation of the endothelium by Ang II promotes aortic stiffness by triggering endothelin-1 release, which in turn acts on aortic smooth muscle cells to induce furin up-regulation, leading to increased activation of MT1-MMP and pro-MMP2 [91]. Conversely, in some contexts, MT1-MMP may down-regulate proteolytic activity, for example by proteolytically processing and inactivating MMP-11 [92]. Both hepsin and matriptase can activate latent pro-urokinase, releasing a potent plasmin-initiated proteolytic cascade that results in the degradation of fibrin and several other ECM proteins, activates MMPs, and can induce the release of growth factors like TGF β, bFGF, and VEGF from the ECM, to enhance endothelial cell migration and invasion [93].

Endothelial protease expression

Expression of genes by endothelial cells may be constitutive or inducible, or may be restricted to subsets of endothelial cell phenotypes in the vascular tree. Endothelial cells have been reported to express mRNAs encoding the membrane-anchored serine proteases testisin, hepsin, matriptase, TMPRSS2, dipeptidylpeptidase IV, and seprase [68]. Our analysis of DataSets in the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geoprofiles) reveals that several membrane-anchored proteases with no known roles in vascular biology are differentially regulated in response to various stimuli. In a study characterizing the endothelial response to laminar shear stress, TMPRSS11A and ADAM18 transcripts were found to be upregulated in response to higher shear stress levels [94]. In a separate study, hepsin transcripts were upregulated in endothelial cells isolated from cirrhotic livers in rats and neonatal murine retinal endothelial cells, which also displayed expression of TMPRSS12 [95] [96]. In another study of retinal angiogenesis utilizing laser capture microdissection of endothelial cell tips, matriptase transcripts were elevated nearly two fold as compared to the surrounding tissue [97]. Investigating transcriptional changes during capillary tube formation on a matrigel basement membrane, ADAM19 transcripts were upregulated over time [98]. TMPRSS2 transcript levels were found to be upregulated when comparing proliferative phase endometrial endothelial cells from polycystic ovary syndrome to normal endometrial endothelial cells [99]. Future studies will be required to analyze proteome expression and determine the pathophysiological functions of membrane-anchored proteases not previously associated with endothelium, in vascular functions and angiogenesis.

Conclusion

The interdependence between protease localization and function provides a mechanism for coordination of extracellular signals and intracellular responses. The membrane-anchored proteases are uniquely positioned at the endothelial plasma membrane to coordinate the release of ECM components, to proteolytically activate cell surface proteins such as PARs, growth factors, and cytokines, to interact with cell surface adhesion receptors and generate soluble ligands, and to communicate with the cytoskeleton and with cellular signaling molecules. While significant progress has been made in understanding the effects of changes in the levels and activities of some of these enzymes, more research is needed on the importance of distinct membrane and subcellular localizations in the control of vascular function under physiological conditions and how pathological states, such as inflammation, change the exposure to substrates that modulate endothelial cell functions. A better understanding of these mechanisms has the potential to provide new insights into perturbations in vascular functions associated with numerous pathophysiological states including cancer, inflammation, aging, neurological diseases, diabetes, atherosclerosis and hypertension while opening up avenues for therapeutic intervention.

Key Points.

• Membrane-anchored proteases are directly linked to the cell surface via Type I or Type II single pass transmembrane domains, or via a glycophosphatidylinositol (GPI)-anchor, with their protease catalytic domains exposed to the extracellular environment.

• Protease function and substrate repertoire can be regulated through localization in distinct areas of the membrane.

• A better understanding of the role of membrane-anchored proteases in vascular biology may provide novel therapeutic targets for the treatment of numerous cardiovascular and angiogenesis related diseases