Endoglin is a transforming growth factor-beta (TGF-beta) binding protein expressed on the surface of endothelial cells. Loss-of-function mutations in the human endoglin gene ENG cause hereditary hemorrhagic telangiectasia (HHT1), a disease characterized by vascular malformations. Here it is shown that by gestational day 11.5, mice lacking endoglin die from defective vascular development. However, in contrast to mice lacking TGF-beta, vasculogenesis was unaffected. Loss of endoglin caused poor vascular smooth muscle development and arrested endothelial remodeling. These results demonstrate that endoglin is essential for angiogenesis and suggest a pathogenic mechanism for HHT1.

Elastin, the main component of the extracellular matrix of arteries, was thought to have a purely structural role. Disruption of elastin was believed to lead to dissection of arteries, but we showed that mutations in one allele encoding elastin cause a human disease in which arteries are blocked, namely, supravalvular aortic stenosis. Here we define the role of elastin in arterial development and disease by generating mice that lack elastin. These mice die of an obstructive arterial disease, which results from subendothelial cell proliferation and reorganization of smooth muscle. These cellular changes are similar to those seen in atherosclerosis. However, lack of elastin is not associated with endothelial damage, thrombosis or inflammation, which occur in models of atherosclerosis. Haemodynamic stress is not associated with arterial obstruction in these mice either, as the disease still occurred in arteries that were isolated in organ culture and therefore not subject to haemodynamic stress. Disruption of elastin is enough to induce subendothelial proliferation of smooth muscle and may contribute to obstructive arterial disease. Thus, elastin has an unanticipated regulatory function during arterial development, controlling proliferation of smooth muscle and stabilizing arterial structure.

Vascular proliferative diseases such as atherosclerosis and coronary restenosis are leading causes of morbidity and mortality in developed nations. Common features associated with these heterogeneous disorders involve phenotypic modulation and subsequent abnormal proliferation and migration of vascular smooth muscle cells into the arterial lumen, leading to neointimal formation and vascular stenosis. This fibrocellular response has largely been attributed to the release of multiple cytokines and growth factors by inflammatory cells. Previously, we demonstrated that the disruption of the elastin matrix leads to defective arterial morphogenesis. Here, we propose that elastin is a potent autocrine regulator of vascular smooth muscle cell activity and that this regulation is important for preventing fibrocellular pathology. Using vascular smooth muscle cells from mice lacking elastin(Eln-/-), we show that elastin induces actin stress fiber organization, inhibits proliferation, regulates migration and signals via a non-integrin, heterotrimeric G-protein-coupled pathway. In a porcine coronary model of restenosis, the therapeutic delivery of exogenous elastin to injured vessels in vivo significantly reduces neointimal formation. These findings indicate that elastin stabilizes the arterial structure by inducing a quiescent contractile state in vascular smooth muscle cells. Together, this work demonstrates that signaling pathways crucial for arterial morphogenesis can play an important role in the pathogenesis and treatment of vascular disease.

The mature circulatory system is comprised of two parallel, yet distinct, vascular networks that carry blood to and from the heart. Studies have suggested that endothelial tubes are specified as arteries and veins at the earliest stages of angiogenesis, before the onset of circulation. To understand the molecular basis for arterial-venous identity, we have focused our studies on a human vascular dysplasia, hereditary haemorrhagic telangiectasia (HHT), wherein arterial and venous beds fail to remain distinct. Genetic studies have demonstrated that HHT can be caused by loss-of-function mutations in the gene encoding activin receptor-like kinase-1 (ACVRL1; ref. 5). ACVRL1 encodes a type I receptor for the TGF-beta superfamily of growth factors. At the earliest stage of vascular development, mice lacking Acvrl1 develop large shunts between arteries and veins, downregulate arterial Efnb2 and fail to confine intravascular haematopoiesis to arteries. These mice die by mid-gestation with severe arteriovenous malformations resulting from fusion of major arteries and veins. The early loss of anatomical, molecular and functional distinctions between arteries and veins indicates that Acvrl1 is required for developing distinct arterial and venous vascular beds.

METHODS Migration AssaysMigration assays were performed as described (3)(4)(5). Briefly, 16 h before the assay, 80% confluent 75 cm 2 flasks (Corning Costar) of human microvessel endothelial cells (HMVEC; Cambrex, Walkersville, MD), human coronary artery endothelial cells (HCAEC; Cambrex), human umbilical artery endothelial cells (HUAEC; Promocell, Heidelburg, Germany), or human umbilical vein endothelial cells (HUVEC; Promocell), were washed with Hank's Balanced Salt Solution (HBSS, Invitrogen) and serum-starved overnight in endothelial basal media (EBM-2, Cambrex) with 0.1% fatty-acid-free BSA (Sigma) and 0.5% fetal calf serum (FCS, Hyclone). The following day cells were lifted with Trypsin/EDTA solution (Promocell), mixed with an equal volume Trypsin Neutralization Solution (Promocell), and washed 3 times in migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS). Cells were resuspended at a density of 1.5×10 6 cells/ml and were allowed to recover for 1 h at 37°C (5% CO 2 ). 3.75 × 10 4 cells were plated into each well of a 48-well Boyden chamber apparatus (NeuroProbe, Cabin John, MD), and the wells were overlayed with an 8 μm pore polycarbonate membrane (NeuroProbe) that had been previously coated with 50 μg/ml human fibronectin (Biomedical Technologies, Inc., Stoughton, MA). Experiments performed with membranes coated with acetylated 1% gelatin from porcine skin (Sigma, St. Louis, MO) gave similar results. The apparatus was assembled and stored inverted at 37°C (5% CO 2 ) for 2 h. The apparatus was then re-inverted and 52 μl of purified chemoattractants [murine netrin-1 (R&D Systems, Minneapolis, MN), chicken netrin-2 (R&D Systems), murine netrin-4 (R&D Systems), murine netrin-G1a (R&D Systems), human VEGF 165 (R&D Systems), or control/ migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS) were added to the upper chambers, and the migration was allowed to proceed for 2 h at 37°C (5% CO 2 ). The membranes were then removed, fixed in methanol, stained with a Hema 3 stain set (Fisher Scientific, Pittsburgh, PA), and placed (migrated-side down) onto 50 × 75 mm glass slides. Before 90% mounting medium (in xylenes) and coverslips were applied, the non-migrated cells were removed from the exposed (non-migrated) side of the membrane with a moistened swab. Cells present on the migrated side of the membrane were manually counted (three random 200× fields per well), and data points for each experiment represent the average number of migrated cells from six separate wells (three 200× fields counted per well).Another method was employed in a separate laboratory to evaluate the effects of the netrins on mouse (MS1) endothelial cells (ATCC, Manassas, VA) using a modified Boyden chamber assay as described previously (6). Briefly, a 5 μm-polycarbonate filter (Poretics) was placed between upper and lower chamber. Cell suspensions (5×10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with serum-free medium containing

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1

Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting. In contrast to known neuronal Robo family members, the arrangement of the extracellular domains of Robo4 diverges significantly from that of all other Robo family members. Moreover, Robo4 is specifically expressed in the vascular endothelium during murine embryonic development. We show that Robo4 binds Slit and inhibits cellular migration in a heterologous expression system, analogous to the role of known Robo receptors in the nervous system. Immunoprecipitation studies indicate that Robo4 binds to Mena, a known effector of Robo-Slit signaling. Finally, we show that Robo4 is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. These data demonstrate that Robo4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development.

Slit-Roundabout (Robo) signalling has a well-understood role in axon guidance1 -5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular