Aubrey C. Chan

The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases

Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and central nervous system blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We show that CCM2 regulates endothelial cytoskeletal architecture, cell-to-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. On the basis of our biochemical studies indicating that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice with simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacological treatment of a human vascular dysplasia with a widely available and safe drug.

Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza

Blunting increased vascular permeability caused by an infection-induced cytokine storm with a Slit ligand increased survival in rodent models of sepsis and viral infection. Batten Down the Vascular Hatches Against the Storm An organism under stress from a massive infection or burn reacts strongly to protect itself. Body-wide inflammation is triggered, but this response can have negative effects of its own. These can include a fast heart rate, abnormally high temperature, and a marked discharge of cytokines from the immune system, called a cytokine storm. Death often occurs in these patients, a result of the failure of multiple organs. In mice, London et al. now inhibit one of the consequences of a cytokine storm—leakage from the vasculature into the intercellular space—and can thereby prevent the lethal effects of bacterial and viral infection. Cytokines released during a cytokine storm, including tumor necrosis factor and interleukin-1β, act on the cells of the vascular lining, weakening their junctions and allowing cells and fluid to leak in. Because the resulting edema is one cause of organ failure, London et al. have targeted this process. They show that an abbreviated version of a soluble ligand, called Slit, when added to cultured vascular endothelial cells, can strengthen the cell-cell contacts. Acting by increasing the amount of a cell adhesion protein, vascular endothelial cadherin, on the cell surface, Slit reduced the permeability of the endothelial cell layer. This was also true in whole animals; the authors injected mice with an immunogenic bacterial protein to simulate infection and then measured vascular leakage. They then tested whether Slit could help mice survive a severe infection. Whether infected with gut bacteria or H5N1 flu, treatment of the mice with the abbreviated Slit molecule improved their odds of surviving. Further, the ability of Slit to reduce vascular permeability in the face of a severe infection depended on another signaling molecule, the Robo4 receptor. Sepsis and other illnesses in which a cytokine storm is triggered are difficult to treat effectively. The standard of care is rapid antibiotic administration and supportive treatment of patients, but this is too often ineffective. The approach described here by London et al. may yield another tool to fight the cytokine storm, a way to strengthen the ability of the body to withstand its own assault. 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 influenza. Thus, enhancing the resilience of the host vascular system to the host’s innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity.

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.

Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.

Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

Background— Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results— We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning–based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. Conclusions— By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans.

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

Recent insights into cerebral cavernous malformations: animal models of CCM and the human phenotype.

Cerebral cavernous malformations are common vascular lesions of the central nervous system that predispose to seizures, focal neurologic deficits and potentially fatal hemorrhagic stroke. Human genetic studies have identified three genes associated with the disease and biochemical studies of these proteins have identified interaction partners and possible signaling pathways. A variety of animal models of CCM have been described to help translate the cellular and biochemical insights into a better understanding of disease mechanism. In this minireview, we discuss the contributions of animal models to our growing understanding of the biology of cavernous malformations, including the elucidation of the cellular context of CCM protein actions and the in vivo confirmation of abnormal endothelial cell–cell interactions. Challenges and progress towards developing a faithful model of CCM biology are reviewed.

Lack of CCM1 induces hypersprouting and impairs response to flow

Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

Erratum: Corrigendum: The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases

Betty YFY Tam, Kevin Wei, John S Rudge, Jana Hoffman, Joceyln Holash, Sang-ki Park, Jenny Yuan, Colleen Hefner, Cecile Chartier, Jeng-Shin Lee, Shelly Jiang, Nihar R Nayak, Frans A Kuypers, Lisa Ma, Uma Sundram, Grace Wu, Joseph A Garcia, Stanley L Schrier, Jacquelyn J Maher, Randall S Johnson, George D Yancopoulos, Richard C Mulligan & Calvin J Kuo Nat. Med. 12, 793–800 (2006); published online 25 June 2006; corrected after print 6 April 2009

Erratum: The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases (Nature Medicine (2009) 15 (177-184))

Betty YFY Tam, Kevin Wei, John S Rudge, Jana Hoffman, Joceyln Holash, Sang-ki Park, Jenny Yuan, Colleen Hefner, Cecile Chartier, Jeng-Shin Lee, Shelly Jiang, Nihar R Nayak, Frans A Kuypers, Lisa Ma, Uma Sundram, Grace Wu, Joseph A Garcia, Stanley L Schrier, Jacquelyn J Maher, Randall S Johnson, George D Yancopoulos, Richard C Mulligan & Calvin J Kuo Nat. Med. 12, 793–800 (2006); published online 25 June 2006; corrected after print 6 April 2009