Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis

Blood vessels change their caliber to adapt to the demands of tissues or organs for oxygen and nutrients. This event is mainly organized at the capillary level and requires a size-sensing mechanism. However, the molecular regulatory mechanism involved in caliber size modification in blood vessels is not clear. Here we show that apelin, a protein secreted from endothelial cells under the activation of Tie2 receptor tyrosine kinase on endothelial cells, plays a role in the regulation of caliber size of blood vessel through its cognate receptor APJ, which is expressed on endothelial cells. During early embryogenesis, APJ is expressed on endothelial cells of the new blood vessels sprouted from the dorsal aorta, but not on pre-existing endothelial cells of the dorsal aorta. Apelin-deficient mice showed narrow blood vessels in intersomitic vessels during embryogenesis. Apelin enhanced endothelial cell proliferation in the presence of vascular endothelial growth factor and promoted cell-to-cell aggregation. These results indicated that the apelin/APJ system is involved in the regulation of blood vessel diameter during angiogenesis.     

Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.     

Development of the hemochorial maternal vascular spaces in the placenta through endothelial and vasculogenic mimicry

The maternal vasculature within the placenta in primates and rodents is unique because it is lined by fetal cells of the trophoblast lineage and not by maternal endothelial cells. In addition to trophoblast cells that invade the uterine spiral arteries that bring blood into the placenta, other trophoblast subtypes sit at different levels of the vascular space. In mice, at least five distinct subtypes of trophoblast cells have been identified which engage maternal endothelial cells on the arterial and venous frontiers of the placenta, but which also form the channel-like spaces within it through a process analogous to formation of blood vessels (vasculogenic mimicry). These cells are all large, post-mitotic trophoblast giant cells. In addition to assuming endothelial cell-like characteristics (endothelial mimicry), they produce dozens of different hormones that are thought to regulate local and systemic maternal adaptations to pregnancy. Recent work has identified distinct molecular pathways in mice that regulate the morphogenesis of trophoblast cells on the arterial and venous sides of the vascular circuit that may be analogous to specification of arterial and venous endothelial cells.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

Correlation of endothelial vimentin content with hemodynamic parameters

 In mammalian species, vimentin is the sole intermediate filament protein of endothelial cells lining the chambers of the heart and the inner surface of large blood vessels. Obvious quantitative differences in the vimentin-like immunoreactivity of endothelial cells observed in different vascular segments led us to undertake a systematic survey on the endothelial content of vimentin throughout the heart chambers, the vena cava, the pulmonary trunk, and the aorta of the pig. Immunostaining and immunoblotting showed that vimentin in endothelial cells of cardiovascular segments exposed to high shear stress and blood pressure (pulmonary trunk, aorta, left ventricle) is approximately 2- to -3-fold higher than in endothelial cells exposed to lower levels of hemodynamic stress (vena cava, left and right atria, right ventricle). Throughout the aorta, an approximately 1.5-fold increase in the vimentin contents was observed in a proximal to distal direction. The total endothelial amount of vimentin was determined to be 1.2% (inferior vena cava) and 2–3.5% (aorta) of total cellular protein. These data support the notion that the endothelial vimentin cytoskeleton can adapt to different hemodynamic loads, indicating that vimentin might help endothelial cells to withstand the mechanical forces exerted by blood flow and blood pressure. Accepted: 2 February 1998     

cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression.

Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma. Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000). Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library. Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs. No significant sequence similarity was observed on analysis of current GenBank databases. The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody. Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein. This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin. Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function. Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney. On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons. Its tissue-specific expression may suggest a functional role in hematopoiesis.     

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions,but Not Blood Pressure,of Ace-/- Mice

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.     

CD105 antagonizes the inhibitory signaling of transforming growth factor beta1 on human vascular endothelial cells.

CD105 (endoglin), a receptor for transforming growth factor beta (TGFbeta), is highly expressed in tissue-cultured, activated endothelial cells in vitro and in tissues undergoing angiogenesis in vivo. The absence of CD105 in knockout mice leads to their death from defective vascular development, but the role of CD105 in the modulation of angiogenesis has not been elucidated. TGFbeta1 is a well-recognized regulator of angiogenesis. Using an antisense approach, we have shown that inhibition of CD105 protein translation in cultured human endothelial cells enhances the ability of TGFbeta1 to suppress growth and migration in these cells. The ability of endothelial cells to form capillary tubes was evaluated by the use of a 3-dimensional collagen matrix system where TGFbeta1 not only reduced the length of capillary-like structures, but also caused massive mortality in CD105-deficient cells compared to control cultures. These results provide direct evidence that CD105 antagonizes the inhibitory effects of TGFbeta1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.     

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.     

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood-brain barrier (BBB)-forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood-cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio-allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio-allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.     

Nestin expression in pancreatic stellate cells and angiogenic endothelial cells

Nestin is an intermediate filament protein expressed by neuroepithelial stem cells and which has been proposed to represent also a marker for putative islet stem cells. The aim of this study was to characterize the cell type(s) expressing nestin in the rat pancreas. By immunohistochemistry, nestin positivity was localized exclusively in mesenchymal cells of normal and regenerating adult pancreas. In the latter condition, the number of nestin-positive cells and the intensity of nestin immunoreactivity were greatly increased. Most nestin-positive cells had the morphology of stellate cells, a type of pericyte associated with blood vessels which has been previously reported to occur in liver and pancreas. In addition, nestin positivity was present in endothelial cells from neocapillaries during pancreas regeneration, and in all blood vessels during morphogenesis in fetal pancreas. Nestin expression was not found in the ductal epithelial cells from which islet cells originate in fetal and regenerating pancreas. In primary pancreatic tissue explants, nestin-positive mesenchymal cells rapidly attached to plastic and proliferated. These cells also expressed desmin, vimentin, and glial fibrillary acidic protein which are known to represent stellate cell markers. In summary, nestin in the pancreas is primarily a marker for reactive stellate cells, or pericytes, and endothelial cells during active angiogenesis.     

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.     

Endothelial Targeting of Cowpea Mosaic Virus (CPMV) via Surface Vimentin

Cowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission.     

α-Tocopheryl succinate stabilizes the structure of tumor vessels by inhibiting angiopoietin-2 expression

α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis.     

Neurothelin: an inducible cell surface glycoprotein of blood-brain barrier-specific endothelial cells and distinct neurons

The blood-brain barrier is characterized by still poorly understood barrier and transport functions performed by specialized endothelial cells. Hybridoma technology has been used to identify a protein termed neurothelin that is specific for these endothelial cells. Neurothelin is defined by the species-specific mouse mAb 1W5 raised against lentil-lectin-binding proteins of neural tissue from embryonic chick. In the posthatch chick, neurothelin expression is found on endothelial cells within the brain but not on those of the systemic vascular system. Injection of the monoclonal antibody in vivo leads to labeling of brain capillaries, indicating that the corresponding antigen is expressed on the luminal surface of brain endothelial cells. Transplantation of embryonic mouse brain onto the chick chorioallantoic membrane results in rodent brain vascularization by the avian vascular system. Subsequently, normally mAb 1W5-negative endothelial cells, originating from blood vessels of the chick chorioallantoic membrane, are induced to express neurothelin when they are in contact with mouse neural tissue. In contrast to differentiated brain neurons that do not express neurothelin, neurons of the nonvascularized chick retina synthesize neurothelin. However, neurothelin is not found on retinal ganglion cell axons terminating on 1W5-negative brain cells. 1W5 immunoreactivity was also found in the pigment epithelium that forms the blood-eye barrier. Putting epithelial cells into culture results in concentration of neurothelin at cell-cell contact sites, leaving other cell surface areas devoid of antigen. Therefore, the distribution of neurothelin appears to be regulated by cell-cell interactions. In Western blot analysis, neurothelin was identified as a protein with a molecular mass of approximately 43 kD. The protein bears at least one intramolecular disulfide bridge and sulfated glucuronic acid as well as alpha-D-substituted mannose/glucose moieties. The exclusive neurothelin expression in the posthatch chick on endothelial cells of the central nervous system but not on systemic endothelial cells makes neurothelin a marker specific for blood-brain barrier-forming endothelial cells. The spatiotemporally regulated neurothelin expression in neurons suggests an interaction between vascularization and neuronal differentiation.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Cathepsin proteases have distinct roles in trophoblast function and vascular remodelling

Trophoblast giant cells are instrumental in promoting blood flow towards the mouse embryo by invading the uterine endometrium and remodelling the maternal vasculature. This process involves the degradation of the perivascular smooth muscle layer and the displacement of vascular endothelial cells to form trophoblast-lined blood sinuses. How this vascular remodelling is achieved at the molecular level remains largely elusive. Here, we show that two placenta-specific cathepsins, Cts7 and Cts8, are expressed in distinct but largely overlapping subsets of giant cells that are in direct contact with maternal arteries. We find that Cts8, but not Cts7, has the capacity to mediate loss of smooth muscle alpha-actin and to disintegrate blood vessels. Consequently, conditional ubiquitous overexpression of Cts8 leads to midgestational embryonic lethality caused by severe vascularization defects. In addition, both cathepsins determine trophoblast cell fate by inhibiting the self-renewing capacity of trophoblast stem cells when overexpressed in vitro. Similarly, transgenic overexpression of Cts7 and Cts8 affects trophoblast proliferation and differentiation by prolonging mitotic cell cycle progression and promoting giant cell differentiation, respectively. We also show that the cell cycle effect is directly caused by some proportion of CTS7 localizing to the nucleus, highlighting the emerging functional diversity of these typically lysosomal proteases in distinct intracellular compartments. Our findings provide evidence for the highly specialized functions of closely related cysteine cathepsin proteases in extra-embryonic development, and reinforce their importance for a successful outcome of pregnancy.