Background:The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. Methods:High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. Results:BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. Conclusions:Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

Background: Kelussia odoratissima (KO) and Angelica sinensis (AS) have been used in their indigenous traditional medicine, for various diseases. This study was conducted to evaluate the volatile oil composition of KO leaves (KVL) and AS root (AVR) and biological activity of essential oils (EOs) and hydroalcoholic extracts of both plants using two different transgenic zebrafish (Danio rerio) models.Materials and Methods: Both EOs were isolated by hydrodistillation and analysed by GC and GC/MS. For viability tests, larvae were treated with different concentrations of extracts to determine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a dose-dependent manner for their biological activity using tissue-specific transgenic zebrafish Tg(fli-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups.Results: Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7% of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that both EOs blocked ISVs formation in the Tg (fli-1: EGFP) embryos increased to 10% of the control value, while both hydroalcoholic extracts did not show any anti-angiogenesis effects in these embryos. In addition, AVR has been shown to significantly induce PBC regeneration following ablation in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5′-N-ethylcarboxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity of both EOs could be attributed to the phthalide structures while for the PBC regenerative activity, other compounds including β-Thujaplicinol, exclusively existing in AVR, might be effective.Conclusion: Although the genera, organs, and origin of these plants are different, their similar chemical composition and biological activities make them valuable resources for further investigation in basic medical and pharmaceutical science.

Hereditary hemorrhagic telangiectasia (HHT) is associated with defective capillary network, leading to dilated superficial vessels and arteriovenous malformations (AVMs) in which arteries connect directly to the veins. Loss or haploinsufficiency of components of TGF-β signaling, ALK1, ENG, SMAD4, and BMP9, have been implicated in the pathogenesis AVMs. Emerging evidence suggests that the inability of endothelial cells to detect, transduce and respond to blood flow, during early development, is an underpinning of AVM pathogenesis. Therefore, components of endothelial flow detection may be instrumental in potentiating TGF-β signaling in perfused blood vessels. Here, we argue that endothelial cilium, a microtubule-based and flow-sensitive organelle, serves as a signaling hub by coupling early flow detection with potentiation of the canonical TGF-β signaling in nascent endothelial cells. Emerging evidence from animal models suggest a role for primary cilia in mediating vascular development. We reason, on recent observations, that endothelial cilia are crucial for vascular development and that embryonic loss of endothelial cilia will curtail TGF-β signaling, leading to associated defects in arteriovenous development and impaired vascular stability. Loss or dysfunction of endothelial primary cilia may be implicated in the genesis of AVMs due, in part, to inhibition of ALK1/SMAD4 signaling. We speculate that AVMs constitute part of the increasing spectrum of ciliopathy-associated vascular defects.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a class of capillary anomalies that are associated with arteriovenous malformations and arteriovenous fistulas, which carry a risk of hemorrhages. There are no broadly effective pharmacological therapies currently available. Most CM-AVMs are associated with a loss of RASA1, resulting in constitutive activation of RAS signaling. However, protein interaction analysis revealed that RASA1 forms a complex with Rho GTPase-activating protein (RhoGAP), a negative regulator of RhoA signaling. Herein, we propose that loss of RASA1 function results in constitutive activation of RhoA signaling in endothelial cells, resulting in enhanced vascular permeability. Therefore, strategies aimed at curtailing RhoA activity should be tested as an adjunctive therapeutic approach in cell culture studies and animal models of RASA1 deficiency.

Skeletal muscle injuries are one of the most common problems in the worldwide which impose a substantial financial burden to the health care system. Accordingly, it widely accepted that muscle regeneration is a promising approach that can be used to treat muscle injury patients. However, the underlying mechanisms of muscle regeneration have yet to be elucidated. The muscle structure and muscle-related gene expression are highly conserved between human and zebrafish. Therefore, the zebrafish can be considered as an ideal animal model in muscle regeneration studies. In this study, Tol2 transposase was applied to produce Tg(mylpfa: cfp-nfsB) zebrafish model that express a fusion protein composed of cyan fluorescent protein (CFP) and nitrorudactase (NTR) under control of mylpfa promoter. The results showed that MTZ (Metronidazole) treatment of Tg(mylpfa:cfp-nfsB) zebrafish larvae can lead to muscle injury by selective ablation of muscle cells. And also, results confirmed the muscle regeneration ability of the transgenic larvae after withdrawal of Mtz for three days. Overall, The results of this study suggest that the Tg(mylpfa:cfp-nfsB) zebrafish model can be used in muscle regeneration study in order to elucidate the mechanisms of this process.

[This corrects the article DOI: 10.3389/fcell.2018.00014.].

Objective— Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell–specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1’s differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus— tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish—we attempted to identify the mechanism of this regulation. Approach and Results— Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein—embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)—that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)–mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide–injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. Conclusions— We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS , and Elavl1 (an interactor of tie1AS ).

Objective— Endothelial cells (ECs) sense and respond to flow-induced mechanical stress, in part, via microtubule-based projections called primary cilia. However, many critical steps during vascular morphogenesis occur independent of flow. The involvement of cilia in regulating these stages of cranial vascular morphogenesis is poorly understood because cilia have not been visualized in primary head vessels. The objective of this study was to investigate involvement of cilia in regulating the early stages of cranial vascular morphogenesis. Approach and Results— Using high-resolution imaging of the Tg(kdrl:mCherry-CAAX ) y171 ;(bactin::Arl13b:GFP ) zebrafish line, we showed that cilia are enriched in the earliest formed cranial vessels that assemble via vasculogenesis and in angiogenic hindbrain capillaries. Cilia were more prevalent around the boundaries of putative intravascular spaces in primary and angiogenic vessels. Loss of cardiac contractility and blood flow, because of knockdown of cardiac troponin T type 2a ( tnnt2a ) expression, did not affect the distribution of cilia in primary head vasculature. In later stages of development, cilia were detected in retinal vasculature, areas of high curvature, vessel bifurcation points, and during vessel anastomosis. Loss of genes crucial for cilia biogenesis ( ift172 and ift81 ) induced intracerebral hemorrhages in an EC-autonomous manner. Exposure to high shear stress induced premature cilia disassembly in brain ECs and was associated with intracerebral hemorrhages. Conclusions— Our study suggests a functional role for cilia in brain ECs, which is associated with the emergence and remodeling of the primary cranial vasculature. This cilia function is flow-independent, and cilia in ECs are required for cerebral-vascular stability.

The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H2S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.

Signaling in pluripotent stem cells is a complex and dynamic process involving multiple mediators, finely tuned to balancing pluripotency and differentiation states. Characterizing and modifying the necessary signaling pathways to attain desired cell types is required for stem-cell applications in various fields of regenerative medicine. These signals may help enhance the differentiation potential of pluripotent cells towards each of the embryonic lineages and enable us to achieve pure in vitro cultures of various cell types. This review provides a timely synthesis of recent advances into how maintenance of pluripotency in hPSCs is regulated by extrinsic cues, such as the fibroblast growth factor (FGF) and ACTIVIN signaling pathways, their interplay with other signaling pathways, namely, wingless-type MMTV integration site family (WNT) and mammalian target of rapamycin (mTOR), and the pathways governing the determination of multiple lineages.

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models is embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.

Dear editor, Intracerebral hemorrhage (ICH) is a severe stroke subtype for which treatment and management options are limited and remain ineffectual at improving the outcome. The question of whether high-dose statin therapy can increase the risk of ICH is a contentious one. Although some clinicians have disputed the link between high-dose statin therapy and the risk of ICH (based on randomized and observational studies), the potential effects of statins on cerebral-vascular stability must not be ignored. Hence, a testable hypothesis is long overdue. It is important to note that statins, in addition to lowering cholesterol levels (both LDLcholesterol and de novo cholesterol synthesis), also inhibit the biosynthesis of other metabolites, namely, the long hydrocarbon lipids, known as prenyl lipids. Prenyl lipids serve as lipid attachments for posttranslational modification and membrane localization of small GTP-binding proteins (GTPases), such as CDC42 and Rac1, both of which play indispensable roles in the maintenance of vascular permeability. In fact, studies in animal models have shown that mutations in genes regulating CDC42 and Rac1activities induce loss of endothelial barrier integrity and smooth muscle cell coverage, resulting in intracerebral hemorrhages. Similarly, we have shown that statin treatment induces cerebral hemorrhages in animal studies through a mechanism that appears to be dependent on prenylation-mediated processes. The extent to which these studies relate to the clinic remains elusive. However, in the absence of a testable hypothesis linking statin treatment with high ICH incidence in population-based clinical findings, we have to rely on the theoretical considerations derived from in-vitro/in-vivo evidence, which clearly suggests that statin treatment can exert deleterious effects on cerebral-vascular stability.

Intracerebral hemorrhage (ICH) is the most severe subtype of stroke. Treatment options are scarce and given the high morbidity and mortality, relatively ineffective. Since patients with ICH may have an unknown heritable component, the need to identify potential risk factors necessitates the use of animal models to elucidate the genetic underpinnings of neurovascular development and, thereby, identify candidate regulatory pathways that are likely to be disrupted in patients with ICH. Zebrafish (Danio rerio) exhibits the anatomical and physiological complexity of a closed circulatory system observed in all vertebrates (with arteries, veins and capillaries). Moreover, studies over the last decade, aided by the application of chemical mutagenesis screens, morpholino mediated knockdown approaches and tissue-specific transgenic markers, have paved the way for the identification of several genes and signaling pathways that regulate developmental neurovascular stabilization. We hypothesize that mutations in these genes or pharmacological perturbations of these gene-products may account, at least in part, for the etiology of some forms of spontaneous ICH in humans.

In a recent publication, Tapia Pérez et al 1 Tapia Pérez J.H. Yildiz O.C. Schneider T. et al. Meta-analysis of statin use for the acute therapy of spontaneous intracerebral hemorrhage. J Stroke Cerebrovasc Dis. 2015; 24: 2521-2526 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar have argued, based on some of the previous meta-analyses, that the risk for statin-induced intracerebral hemorrhage (ICH) has been refuted. However, this is a premature and somewhat misleading conclusion, as whether high-dose statin therapy can elicit ICH remains to be a subject of contention among researchers and clinicians. 2 MacDonald R.L. Are statins to be STACHed in subarachnoid haemorrhage?. Lancet Neurol. 2014; 13: 639-641 Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar In fact, according to the most recent guidelines by the American Stroke Association for the management of ICH, the question of whether statin therapy should be continued in patients remains elusive. 3 Hemphill 3rd, J.C. Greenberg S.M. Anderson C.S. et al. Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2015; 46: 2032-2060 Crossref PubMed Scopus (1961) Google Scholar Furthermore, Tapia Pérez et al 1 Tapia Pérez J.H. Yildiz O.C. Schneider T. et al. Meta-analysis of statin use for the acute therapy of spontaneous intracerebral hemorrhage. J Stroke Cerebrovasc Dis. 2015; 24: 2521-2526 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar have suggested that statins may exert neuroprotective effects in ICH patients through cholesterol-independent mechanisms, more specifically, via modulation of small GTPases (through inhibition of prenylation). Although this is a valid point, the authors have failed to explain, mechanistically, how statin-induced modulation of small GTPase activities can also disrupt the permeability of the vascular endothelium. It is important to note that some of these small GTPases, chief among them Rac1 or CDC42, are required for the maintenance of cerebral–vascular permeability. 4 Birukov K.G. Bochkov V.N. Birukova A.A. et al. Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac. Circ Res. 2004; 95: 892-901 Crossref PubMed Scopus (136) Google Scholar , 5 Kouklis P. Konstantoulaki M. Vogel S. et al. Cdc42 regulates the restoration of endothelial barrier function. Circ Res. 2004; 94: 159-166 Crossref PubMed Scopus (115) Google Scholar , 6 Broman M.T. Kouklis P. Gao X. et al. Cdc42 regulates adherens junction stability and endothelial permeability by inducing alpha-catenin interaction with the vascular endothelial cadherin complex. Circ Res. 2006; 98: 73-80 Crossref PubMed Scopus (78) Google Scholar , 7 Koh W. Mahan R.D. Davis G.E. Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling. J Cell Sci. 2008; 121: 989-1001 Crossref PubMed Scopus (160) Google Scholar In fact, downregulation of Rac1/CDC42 activities is a hallmark of cerebral–vascular disorders such as cerebral cavernous malformations, which are usually associated with microvascular leakage and, in severe case, cerebral hemorrhages. 8 Faurobert E. Albiges-Rizo C. Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction. FEBS J. 2010; 277: 1084-1096 Crossref PubMed Scopus (84) Google Scholar Consistently, we have recently shown in animal studies that statin treatment induces ICH by inhibiting the prenylation (at the CAAX terminus) of the small GTPases, Rac1, and CDC42. 9 Eisa-Beygi S. Hatch G. Noble S. et al. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signalling pathway. Dev Biol. 2013; 373: 258-266 Crossref PubMed Scopus (43) Google Scholar , 10 Eisa-Beygi S. Wen X.Y. Macdonald R.L. A call for rigorous study of statins in resolution of cerebral cavernous malformation pathology. Stroke. 2014; 45: 1859-1861 Crossref PubMed Scopus (21) Google Scholar Hence, the potential cholesterol-independent (pleiotropic) effects of statins on cerebral–vascular architecture need to be evaluated mechanistically before any conclusive statements (based only on meta-analyses) are made regarding the putative mechanisms and the risk/benefit of statins in patients with ICH.

Activation of the RhoA/Rho-kinase (ROCK) pathway induces endothelial barrier dysfunction and increased vascular permeability, which is a hallmark of various life-threatening vascular pathologies. Therapeutic approaches aimed at inhibiting the RhoA/ROCK pathway have proven effective in the attenuation of vascular leakage observed in animal models of endotoxin-induced lung injury/sepsis, edema, autoimmune disorders, and stroke. These findings suggest that treatments targeting the ROCK pathway might be of benefit in the management of the Ebola virus disease (EVD), which is characterized by severe vascular leak, likely involving pro-inflammatory cytokines, such as tumor necrosis factor-alpha, released from virus-infected macrophages. In this paper, we review evidence from in vivo and in vitro models of vascular leakage, suggesting that the RhoA/ROCK pathway is an important therapeutic target for the reversal of the vascular permeability defects associated with EVD. Future studies should explore the efficacy of pharmacological inhibition of RhoA/ROCK pathway on reversing the endothelial barrier dysfunction in animal models of EVD and other hemorrhagic fever virus infections as part of an adjunctive therapy. Such experimental studies should focus, in particular, on the small molecule fasudil (HA-1077), a derivative of isoquinoline, which is a safe and clinically approved inhibitor of ROCK, making it an excellent candidate in this context.

HomeStrokeVol. 45, No. 6A Call for Rigorous Study of Statins in Resolution of Cerebral Cavernous Malformation Pathology Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBA Call for Rigorous Study of Statins in Resolution of Cerebral Cavernous Malformation Pathology Shahram Eisa-Beygi, PhD, Xiao-Yan Wen, MD, PhD and R. Loch Macdonald, MD, PhD Shahram Eisa-BeygiShahram Eisa-Beygi From the Zebrafish Centre for Advanced Drug Discovery (S.E.-B., X.-Y.W., R.L.M.) and Keenan Research Centre for Biomedical Science (S.E.-B., X.-Y.W., R.L.M.), St. Michael's Hospital, Toronto, Ontario, Canada; Departments of Medicine and Surgery, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada (S.E.-B., X.-Y.W., R.L.M.); and Division of Neurosurgery, St. Michael's Hospital, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Toronto, Ontario, Canada (R.L.M.). , Xiao-Yan WenXiao-Yan Wen From the Zebrafish Centre for Advanced Drug Discovery (S.E.-B., X.-Y.W., R.L.M.) and Keenan Research Centre for Biomedical Science (S.E.-B., X.-Y.W., R.L.M.), St. Michael's Hospital, Toronto, Ontario, Canada; Departments of Medicine and Surgery, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada (S.E.-B., X.-Y.W., R.L.M.); and Division of Neurosurgery, St. Michael's Hospital, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Toronto, Ontario, Canada (R.L.M.). and R. Loch MacdonaldR. Loch Macdonald From the Zebrafish Centre for Advanced Drug Discovery (S.E.-B., X.-Y.W., R.L.M.) and Keenan Research Centre for Biomedical Science (S.E.-B., X.-Y.W., R.L.M.), St. Michael's Hospital, Toronto, Ontario, Canada; Departments of Medicine and Surgery, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada (S.E.-B., X.-Y.W., R.L.M.); and Division of Neurosurgery, St. Michael's Hospital, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Toronto, Ontario, Canada (R.L.M.). Originally published6 May 2014https://doi.org/10.1161/STROKEAHA.114.005132Stroke. 2014;45:1859–1861Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2014: Previous Version 1 IntroductionCerebral cavernous malformations (CCMs) are cerebrovascular disorders with an approximate prevalence of 1 in 200. The CCM pathology is typified by abnormally dilated clusters of blood vessels with defective endothelial cell–cell junctions, sluggish blood flow, and almost always associated with hemosiderin deposition in the surrounding parenchyma.1–3 CCM can arise sporadically or be inherited in an autosomal dominant pattern.4–6 In some cases, CCMs cause hemorrhagic stroke that elicits neurological defects and rarely death. Extravasataion of blood components and hematoma expansion can often occur asymptomatically. Three structurally unrelated genes, CCM1 (KRIT1), CCM2 (MGC4607), and CCM3 (PDCD10), have been implicated in CCM pathobiology.7–9 It is now thought that the CCM proteins form a ternary complex near the plasma membrane of endothelial cells and act as scaffolds linking the junctional proteins, integrins and vascular endothelial–cadherin, with intracellular signaling components.10 However, the specific mechanisms through which reductions in the expression of the structurally diverse genes associated with CCM induce their formation and pathobiology are not well explained. Studies using mouse and zebrafish models have been instrumental in functionally characterizing and, to an extent, faithfully recapitulating the molecular and ultrastructural underpinnings of CCM pathology.11–15Results from these studies have led to the suggestion that pharmacological inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) by statins may be an effective approach to prevent CCM-induced vascular instability and cerebral hemorrhage.12,16 The basis for this proposition stems from in vivo and in vitro evidence suggesting that loss of function of CCM1, CCM2, or CCM3 genes are associated with RhoA hyperactivation and downstream signaling via Rho kinase and increased stress fiber assembly, which leads to disrupted endothelial cell–cell junctions and loss of vascular stability.17,18 More specifically, activated RhoA, through its effector, Rho kinase, mediates actin stress fiber formation by increasing myosin light chain phosphorylation, as well as inhibiting myosin phosphatase, which is associated with vascular hyperpermeability.19 Consistently, pharmacological curtailment of RhoA activity, using fasudil, a relatively selective RhoA/Rho kinase inhibitor,20 has been shown to enhance vascular stability effectively in vivo and in vitro and significantly reduce the prevalence of CCM lesions in a in Krit1+/− and CCM2+/− mice.17,21Alongside, it has been reported, in ≥1 animal study, that in mice with a heterozygous mutation of CCM2, treatment with statins (simvastatin) effectively restores the endothelial barrier function by inhibiting Rho GTPase activity,12 presumably through abrogating the prenylation process. This experimental finding has led the authors to the conclusion that statin treatment could be an effective alternative to neurosurgical intervention to improve CCM outcome.12,16 However, there are several outstanding questions about the efficacy and specificity of statins as therapeutic agents to improve CCM outcome. First, unlike fasudil, which is a somewhat selective inhibitor of RhoA/Rho kinase,20 statins are competitive inhibitors of HMGCR, the rate-limiting enzyme in the biosynthesis of isoprenoid pyrophosphates, a highly conserved metabolic pathway (Figure).22 These mevalonate derivatives in turn serve as substrates for post-translational modification (prenylation) of a variety of cell signaling proteins (>100) that harbor the C-terminal CaaX motif, the most well characterized of which include the small GTPase family of molecular switch proteins, RhoA, Rac1, and Cdc42 (Figure).23,24 The CaaX proteins interact with prenyltransferases that modify the CaaX cysteine residue by forming a thioether linkage with either a farnesyl or a geranylgeranyl lipid moiety, which ensures membrane localization.25 Hence, inhibition of HMGCR not only reduces the availability of prenyl-based metabolites but also curtails the activity of key cell signaling molecules that require prenylation for activation and downstream signaling.Download figureDownload PowerPointFigure. 3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)–dependent regulation of Cdc42/Rac1 GTPase prenylation and βPix/PAK–mediated signal transduction pathway involved in actin filament stabilization. FPP indicates farnesyl pyrophosphate; GAP, GTPase activating protein; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl diphosphate; LIMK, LIM kinase; and PAK, p21 activated kinase.Therefore, a point of contention about statin therapy is whether inhibition of RhoA hyperactivity is outweighed by any potential pathological outcomes associated with general or indiscriminate depletion of all prenylation-dependent cellular processes. Although statins are generally well tolerated in patients, with rare medically significant side effects, little is known about the possible complications of impaired HMGCR function on the endothelium. For example, prenylated and GTP-bound cdc42/Rac1 plays vital roles in the regulation of vacuole formation, lumenization of vessels, and mediation of endothelial barrier function through regulating vascular endothelial–cadherin dynamics.26–31 Consistently, studies in vivo in zebrafish have shown that genetic depletion of βPix, a guanine nucleotide exchange factor involved in activating Cdc42/Rac1 (by increasing affinity for GTP), as well as p21-activated kinase, a kinase acting downstream of Cdc42/Rac1 (Figure), disrupt vascular integrity and is associated with cerebral hemorrhages and defective vascular stabilization.32,33 Waterborne exposure to statins (atorvastatin and cerivastatin) and morpholino-mediated depletion of embryonic HMGCR transcripts in zebrafish have been shown to induce intracerebral hemorrhage in both embryos and larvae due likely to impaired prenylation-dependent processes.34–36 Surprisingly, though, there exist no experimental or anecdotal studies to suggest statin-induced enhancement of vascular permeability in mice or other mammalian studies, which could suggest species-specific physiological differences. There have also been no studies, to date, to suggest that statins disrupt vascular stability in murine models of aging, hypertensive and amyloid angiopathies. Nonetheless, in light of the theoretical/mechanistic considerations derived from in vitro and in vivo studies (Figure), we acknowledge that there is a need for a more rigorous set of mammalian studies to investigate the putative molecular mechanisms or the risk benefit of statins in CCM pathology.DisclosuresDr Macdonald receives grant support from the Physicians Services Incorporated Foundation, Brain Aneurysm Foundation, Canadian Institutes for Health Research, and the Heart and Stroke Foundation of Canada and is Chief Scientific Officer of Edge Therapeutics, Inc. The other authors report no conflicts.FootnotesThe opinions expressed in the article are not necessarily those of the editors or of the American Heart Association.Correspondence to Shahram Eisa-Beygi, PhD, Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital, 209 Victoria St, LKSKI, Rm 519, Toronto M5B 1T8, Ontario, Canada. E-mail [email protected]References1. Clatterbuck RE, Eberhart CG, Crain BJ, Rigamonti D. Ultrastructural and immunocytochemical evidence that an incompetent blood-brain barrier is related to the pathophysiology of cavernous malformations.J Neurol Neurosurg Psychiatry. 2001; 71:188–192.CrossrefMedlineGoogle Scholar2. Wang X, Tao Z, You C, Li Q, Liu Y. Extended resection of hemosiderin fringe is better for seizure outcome: a study in patients with cavernous malformation associated with refractory epilepsy.Neurol India. 2013; 61:288–292.CrossrefMedlineGoogle Scholar3. Dalyai RT, Ghobrial G, Awad I, Tjoumakaris S, Gonzalez LF, Dumont AS, et al. 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June 2014Vol 45, Issue 6 Advertisement Article InformationMetrics © 2014 American Heart Association, Inc.https://doi.org/10.1161/STROKEAHA.114.005132PMID: 24803598 Manuscript receivedFebruary 13, 2014Manuscript acceptedApril 9, 2014Originally publishedMay 6, 2014Manuscript revisedApril 7, 2014 Keywordscerebral hemorrhagehemangioma, cavernous, central nervous systemPDF download Advertisement SubjectsCerebral AneurysmCerebrovascular Disease/StrokeIntracranial Hemorrhage

The endothelial-specific transmembrane glycoprotein, vascular endothelial (VE)-cadherin, is required for the organization of a stable vascular endothelium. A number of cerebrovascular disorders are associated with mutations in genes that otherwise regulate vascular integrity through VE-cadherin dynamics. Hence, identification and characterization of regulatory pathways contributing to endothelial cell-cell adhesion is of clinical relevance, particularly in the treatment of aneurysms and cerebral cavernous malformations. The zebrafish ( Danio rerio) have recently emerged as a powerful paradigm for studies geared toward elucidating the etiology of cerebrovascular disorders, principally in uncovering the genetic and mechanistic basis controlling endothelial adhesive barrier function.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, which are substrates required for post-translational modification of signalling proteins that can potentially regulate various aspects of embryonic development. The HMGCR transcripts are detectable during early embryogenesis in both invertebrates and vertebrates, which suggests a conserved developmental requirement for mevalonate derivatives. Consistently, recent animal and in vitro studies have yielded valuable insights into potential morphogenic parameters that are modulated by HMGCR activity. These developmental end-points include brain and craniofacial morphogenesis, PGC migration and survival, myocardial epithelial migration and fusion, EC migration and survival, and vascular stabilization. By providing a synthesis of these studies, we hope that this review will highlight the need to comprehensively examine the entire suite of developmental processes regulated by HMGCR.