The described invention relates to small molecule therapeutic compounds capable of reducing the incidence of intracerebral hemorrhage and brain microhemorrhages identified using zebrafish and mouse models of intracerebral hemorrhage and brain microhemorrhages.

Many pathologic conditions cause a destabilization of the vascular network resulting in endothelial hyperpermeability, excessive vascular sprouting, and angiogenesis. (Smith M C P, Li, D Y, Whitehead, K J (2010) "Mechanisms of vascular stability and the relationship to human disease," Curr. Opin. Hematol. 17(30: 237-44).
Vascular Permeability
Vascular permeability is an extremely complex process that, in different settings, involves distinctly different types of blood vessels and makes use of different anatomic and molecular pathways.
While the vascular system of higher organisms is often described as "closed", it needs to be sufficiently "open" (i.e., "permeable") to allow the ready exchange of small molecules (gases, nutrients, waste products) with the tissues. (Nagy, J. A., Benjamin, L., Zeng, H., Dvorak, A. M., Dvorak, H. F. (2008) "Vascular permeability, vascular hyperpermeability and angiogenesis," Angiogenesis 11(2): 109-119). Plasma proteins also need to cross the normal vascular barrier, at least in small amounts.
Permeability, meaning the net amount of a solute, typically a macromolecule, that has crossed a vascular bed and accumulated in the interstitium in response to a vascular permeabilizing agent or at a site of pathological angiogenesis, is an extremely complicated process that is affected by many different variables. Id. These include the intrinsic properties of the different types of microvessels involved (capillaries, venules, mother vessels (MV)); the size, shape, and charge of extravasating molecules; the anatomic pathways molecules take in crossing the endothelial cell barrier; the time course over which permeability is measured; and the animals and vascular beds that are being investigated. Id.
Basal Vascular Permeability (BVP)
Molecular exchange in normal tissues takes place primarily in capillaries, largely by diffusion. The molecules exchanged consist largely of gases (O2 and CO2), water, small molecules such as salts and sugars, and only small amounts of plasma proteins. Id. The extent of BVP varies considerably in different normal tissues and is subject to substantial change in response to changes in hydrostatic pressure, opening of closed vessels, surface area available for exchange, blood flow, etc. Id.
Water and lipophilic solutes (e.g., gases such as O2 and CO2) are able to diffuse through endothelial cells; they also pass readily through inter-endothelial cell junctions and through endothelial fenestrae. Id. Small lipophilic molecules can also dissolve in endothelial cell membranes and so pass from the vascular lumen to the interstitium. Id. Capillary endothelial cells contain large numbers of small (about 70 nm diameter) vesicles (caveolae) the majority of which are found connected to the luminal and abluminal plasma membranes by means of stomata that are generally closed by thin diaphragms containing plasmalemmal vesicle associated protein (PV-1), an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of vaveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. (Stan, R V, Tkachenko, E., Niesman, I R, (2004), "PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms," Mol. Biol. Cell 15(8): 3615-30). Others are in the cytoplasm.
Acute Vascular Hyperpermeability (AVH)
A rapid increase in vascular permeability occurs when the microvasculature is exposed acutely to any of a number of vascular permeabilizing factors, for example, VEGF-A, histamine, serotonin, PAF, etc. Some of these agents (e.g., histamine, serotonin, VEGF-A) are normally stored in tissue mast cells (Nagy, J. A., Benjamin, L., Zeng, H., Dvorak, A. M., Dvorak, H. F. (2008) "Vascular permeability, vascular hyperpermeability and angiogenesis," Angiogenesis 11(2): 109-119, citing Boesiger J, Tsai M, Maurer M et al (1998) Mast cells can secrete vascular permeability factor/vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of fc epsilon receptor I expression. J Exp Med 188:1135-1145; Galli S J (2000) Mast cells and basophils. Curr Opin Hematol 7:32-39; Galli S J (1997) The Paul Kallos Memorial Lecture. The mast cell: a versatile effector cell for a challenging world. Int Arch Allergy Immunol 113:14-22) and so may be released by agents that cause mast cell degranulation, e.g., allergy, insect bites, etc. Single exposure to any of these permeability factors results in a rapid but self-limited (complete by 20-30 min) influx of plasma into the tissues.
The quantity of extravasated fluid in AVH is greatly increased above that found in BVP and its composition is greatly changed. The fluid that extravasates in AVH is rich in plasma proteins, approaching the levels found in plasma, and is referred to as an exudate. Id. Among the plasma proteins that extravasate are fibrinogen and various members of the blood clotting cascade. Id. When these come into contact with tissue factor, a protein that is normally expressed by many interstitial cells, the clotting system is activated and the exudate clots to deposit fibrin (Id. Citing Dvorak H F, Quay S C, Orenstein N S et al (1981) Tumor shedding and coagulation. Science 212:923-924; VanDeWater L, Tracy P B, Aronson D et al (1985) Tumor cell generation of thrombin via functional prothrombinase assembly. Cancer Res 45:5521-5525). Fibrin forms a gel that traps water and other solutes, restraining their clearance by lymphatics or capillaries and resulting in tissue swelling (edema). Id. As long as the permeability stimulus is not continuous, the deposited fibrin is rapidly degraded without further consequences. Id.
AVH also differs from BVP in that the vascular leakage takes place from post-capillary venules, highly specific vessels just downstream of capillaries (Id. Citing Majno G, Palade G E, Schoefl G I (1961) Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree: a topographic study. J Biophys Biochem Cytol 11:607-626; Majno G, Shea S M, Leventhal M (1969) Endothelial contraction induced by histamine-type mediators: an electron microscopic study. J Cell Biol 42:647-672). It has been proposed that histamine and other vascular permeabilizing agents induce endothelial cells to contract and pull apart to form intercellular (paracellular) gaps of sufficient size to permit plasma-protein extravasation. Id. In addition, venular epithelium contains a structure, the vesiculo-vacuolar organelle (VVO), that offers an alternative, trans-endothelial cell route for plasma extravasation in response to permeability factors (Id. citing Kohn S, Nagy J A, Dvorak H F et al (1992) Pathways of macromolecular tracer transport across venules and small veins. Structural basis for the hyperpermeability of tumor blood vessels. Lab Invest 67:596-607; Dvorak A M, Kohn S, Morgan E S et al (1996) The vesiculo-vacuolar organelle (VVO): a distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation. J Leukoc Biol 59:100-115; Feng D, Nagy J, Dvorak A et al (2000) Different pathways of macromolecule extravasation from hyperpermeable tumor vessels. Microvascular Research 59:24-37; Feng D, Nagy J A, Hipp J et al (1996) Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin. J Exp Med 183:1981-1986; Feng D, Nagy J A, Hipp J et al (1997) Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-pig, mouse and rat: many are transcellular pores. J Physiol 504(Pt 3):747-761). VVOs, which are grape-like clusters comprised of hundreds of uncoated, cytoplasmic vesicles and vacuoles that together form an organelle that traverses venular endothelial cytoplasm, often extend to inter-endothelial cell interfaces and their individual vesicles (unlike caveolae) commonly open to the inter-endothelial cell cleft. Id. The vesicles and vacuoles comprising VVOs vary in size from those the size of caveolae to vacuoles with volumes as much as 10-fold larger (Feng D, Nagy J A, Pyne K et al (1999) Pathways of macromolecular extravasation across microvascular endothelium in response to VPF/VEGF and other vasoactive mediators. Microcirculation 6:23-44). These vesicles and vacuoles are linked to each other and to the luminal and abluminal plasma membranes by stomata that are normally closed by thin diaphragms that appear similar to those found in caveolae. Id. It has been proposed that vascular permeability inducing agents cause the diaphragms interconnecting vesicles and vacuoles to open, thereby providing a transcellular pathway for plasma and plasma-protein extravasation. Id.
Chronic Vascular Hyperpermeability (CVH)
Chronic exposure to permeability factors results in profound changes in venular structure and function that lead to the chronic hyperpermeability of pathological angiogenesis as found in tumors, healing wounds, and chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, cellular immunity, etc. (Id. Citing Dvorak H F (2003) Rous-Whipple award lecture. How tumors make bad blood vessels and stroma. Am J Pathol 162:1747-1757; Nagy J A, Masse E M, Herzberg K T et al (1995) Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability, and ascites fluid accumulation. Cancer Res 55:360-368). As in AVH, the fluid that extravasates is an exudate that approaches the overall composition of plasma. In tumors fluid accumulation is generally associated with increased interstitial pressure (Id. Citing Jain R K (1988) Determinants of tumor blood flow: a review. Cancer Res 48:2641-2658); this increased pressure results from persistent vascular hyperpermeability, clotting of the exudate with deposition of a fluid-trapping fibrin gel, inadequate lymphatic drainage, and the restraints imposed by surrounding tissues that together limit fluid dissipation. Id. These restraints are nearly absent when tumors grow in or around body cavities such as the peritoneum where massive amounts of ascites fluid can accumulate. Id.
In contrast to BVP and AVH, fluid leakage in CVH does not take place from any type of normal blood vessel. Instead, whether in tumors or wounds, the blood vessels that leak are newly formed, angiogenic blood vessels; these are primarily mother vessels (MV), and also, to a lesser extent, glomeruloid microvascular proliferations (GMP) that form from MV (Id. Citing Nagy J A, Feng D, Vasile E et al (2006) Permeability properties of tumor surrogate blood vessels induced by VEGF-A. Lab Invest 86:767-780; Pettersson A, Nagy J A, Brown L F et al (2000) Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab Invest 80:99-115; Sundberg C, Nagy J A, Brown L F et al (2001) Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery. Am J Pathol 158:1145-1160; Brown L F, Detmar M, Claffey K et al (1997) Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine. Exs 79:233-269; Brown L F, Yeo K T, Berse B et al (1992) Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 176:1375-1379; Ren G, Michael L H, Entman M L et al (2002) Morphological characteristics of the microvasculature in healing myocardial infarcts. J Histochem Cytochem 50:71-79. Mother Vessels are greatly enlarged sinusoids that arise from preexisting normal venules by a process that involves pericyte detachment, vascular basal lamina degradation, and a 4-5-fold increase in lumen size that is accompanied by extensive endothelial cell thinning. Id. Notwithstanding that Poiseuille's law indicates that blood flow (flow rate) is proportional to the fourth power of the vascular radius, MV exhibit sluggish blood flow because of their hyperpermeability to plasma which results in a striking increase in hematocrit. Id. The protein-rich exudates in CVH interact with tissue factor to trigger the clotting system and deposit fibrin (Id. Citing Dvorak H F, Quay S C, Orenstein N S et al (1981) Tumor shedding and coagulation. Science 212:923-924; VanDeWater L, Tracy P B, Aronson D et al (1985) Tumor cell generation of thrombin via functional prothrombinase assembly. Cancer Res 45:5521-5525).
Tissue factor is expressed on many tumor cells as well as host interstitial cells and is induced in endothelial cells by VEGF-A (Id). In addition to its fluid trapping properties, fibrin also has a number of other properties when it persists over time as in tumors and healing wounds. It provides a pro-angiogenic provisional stroma that induces and is later replaced by the ingrowth of new blood vessels and fibroblasts and the laying down of mature fibro-vascular stroma (Id. Citing Dvorak H F (2003) Rous-Whipple award lecture. How tumors make bad blood vessels and stroma. Am J Pathol 162:1747-1757; Dvorak H F, Orenstein N S, Carvalho A C et al (1979) Induction of a fibrin-gel investment: an early event in line 10 hepatocarcinoma growth mediated by tumor-secreted products. J Immunol 122:166-174; Dvorak H F, Dvorak A M, Manseau E J et al (1979) Fibrin gel investment associated with line 1 and line 10 solid tumor growth, angiogenesis, and fibroplasia in guinea pigs. Role of cellular immunity, myofibroblasts, microvascular damage, and infarction in line 1 tumor regression. J Natl Cancer Inst 62:1459-1472). Fibrin interacts with integrins expressed by multiple cell types, thereby supporting the migration of tumor cells as well as host mesenchymal cells (endothelial cells, pericytes, fibroblasts) and inflammatory cells (neutrophils, monocytes). Id. Fibrin also sequesters growth factors, protecting them from degradation, and induces the expression of proangiogenic molecules such as IL-8 and tissue factor. Id. Fragment E, a fibrin breakdown product, is directly pro-angiogenic (Id.). Macromolecules extravasate from MV and GMP largely via a transcellular route (Id. Citing Nagy J A, Feng D, Vasile E et al (2006) Permeability properties of tumor surrogate blood vessels induced by VEGF-A. Lab Invest 86:767-780).
In short, while agents such as VEGF-A have long been known to induce AVH and CVH, apart from hemodynamic factors, much less is known about the molecular events that are responsible for the normal permeability of BVP, and even less is known about the molecules that are involved in regulating permeability, and the molecular mechanisms that govern each of the different types of permeability may well be different. The signaling pathways by which even such well-studied molecules as eNOS and caveolin-1 act to induce permeability are poorly understood. Id. Little is known about the molecular mechanisms that regulate such critical events as caveolar shuttling, the opening of VVO diaphragms, the formation of fenestrae, changes in endothelial cell junctions, etc. (Id. Citing Dejana E (2004) Endothelial cell-cell junctions: happy together. Nat Rev Mol Cell Biol 5:261-270; Oh P, Borgstrom P, Witkiewicz H et al (2007) Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechno125:327-337; Ioannidou S, Deinhardt K, Miotla J et al (2006) An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis. Proc Natl Acad Sci USA 103:16770-16775).
Angiogenesis
Angiogenesis is a process of neovascular formation from pre-existing blood vessels during embryogenesis, adult tissue homeostasis and carcinogenesis. (Katoh, M., (2013) "Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks," Intl J. Mol. Med. 32(4): 763-67, citing Carmeliet P. (2005) Angiogenesis in life, disease and medicine. Nature. 438:932-936; Ferrara N, Kerbel R S. (2005) Angiogenesis as a therapeutic target. Nature. 438:967-974; Folkman J. (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 6:273-286; Carmeliet P, Jain R K. (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature. 473:298-307). It is distinct from vasculogenesis which is the developmental in situ differentiation and growth of blood vessels from mesodermal derived hemangioblasts.
Angiogenesis occurs in multiple steps as follows: i) vascular destabilization induced by degradation of the basement membrane and decreased adhesion of endothelial cells; ii) angiogenic sprouting resulting from the migration of endothelial tip cells and the proliferation of endothelial stalk cells; iii) lumen formation by endothelial cells and the recruitment of pericytes to the surrounding region of the endothelial lumen; iv) vascular stabilization depending on tight junctions and basement membrane. (Katoh, M., (2013) "Therapeutics targeting angiogenesis—genetics and epigenetics, extracellular miRNAs and signaling networks," Intl J. Mol. Med. 32(4): 763-67, citing Carmeliet P. (2005) Angiogenesis in life, disease and medicine. Nature. 438:932-936).
Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2), angiopoietins (ANGPT1 and ANGPT2), Notch ligands [jagged 1 (JAG1) and Delta like ligand 4 (DLL4)] and transforming growth factor-β (TGF-β) regulate angiogenesis through their receptors on vascular endothelial cells. VEGF activates the endothelial nitric acid oxide synthase (eNOS), SRC, RAS-ERK and PI3K-AKT signaling cascades through VEGFR2 receptor on endothelial cells, which induce vascular permeability, endothelial migration, proliferation and survival, respectively (Id. Citing Coultas L, Chawengsaksophak K, Rossant J. (2005) "Endothelial cells and VEGF in vascular development." Nature. 438:937-945; Olsson A K, Dimberg A, Kreuger J, Claesson-Welsh L. (2006) "VEGF receptor signaling-in control of vascular function." Nat Rev Mol Cell Biol. 7:359-371). FGF2 promotes angiogenesis directly through FGFR1 receptor on endothelial cells via signaling cascades similar to VEGF, or indirectly through VEGF secretion from endothelial cells, cardiomyocytes and stromal cells (Id. Citing Presta M, Dell'Era P, Mitola S, et al. (2005) "Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis." Cytokine Growth Factor Rev. 16:159-178). ANGPT1, secreted from pericytes, activates TEK/TIE2 receptor to maintain endothelial quiescence or stabilization, whereas ANGPT2, secreted from endothelial cells themselves by VEGF or hypoxia signaling, inhibits TEK to promote endothelial activation or sprouting (Id. Citing Fagiani E, Christofori G. (2013) "Angiopoietins in angiogenesis." Cancer Lett. 328:18-26). JAG1-Notch signaling promotes angiogenic sprouting, whereas DLL4-Notch signaling inhibits angiogenic sprouting (Id. Citing Bridges E, Oon C E, Harris A. (2011) "Notch regulation of tumor angiogenesis." Future Oncol. 7:569-588). TGF-β signaling through TGFBR1/ALK5 receptor to the Smad2/3 cascade inhibits endothelial cell activation, maintaining endothelial quiescence, whereas TGF-β signaling through the ACVRL1/ALK1 receptor to the Smad1/5 cascade promotes the migration and proliferation of endothelial cells (Id. Citing Gaengel K, Genové G, Armulik A, Betsholtz C. (2009) "Endothelial-mural cell signaling in vascular development and angiogenesis." Arterioscler Thromb Vasc Biol. 29:630-638). The VEGF, FGF, Notch and TGF-β signaling cascades are directly involved in the angiogenic signaling of endothelial cells (Id).
The VEGF, FGF, Notch and TGF-β signaling cascades cross-talk with WNT and Hedgehog signaling cascades to constitute the stem-cell signaling network (Id. Citing Katoh M, Katoh M. (2007) "WNT signaling pathway and stem cell signaling network." Clin Cancer Res. 13:4042-4045; Katoh Y, Katoh M. (2008) "Hedgehog signaling, epithelial-to-mesenchymal transition and miRNA," Int J Mol Med. 22:271-275). DVL2-binding deubiquitinase FAM105B regulates WNT signaling and angiogenesis (Id. Citing Rivkin E, Almeida S M, Ceccarelli D F, et al. (2013) "The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis." Nature 498:318-324), while Hedgehog signaling is involved in the regulation of liver sinusoidal endothelial cells (Id. Citing Diehl A M. (2012) "Neighborhood watch orchestrates liver regeneration." Nat Med. 18:497-499). FGF, Notch and canonical WNT signaling are involved in cell-fate determination based on mutual transcriptional regulation, whereas FGF, Notch, TGF-β, Hedgehog and non-canonical WNT signaling are involved in epithelial-to-mesenchymal transition (EMT) due to the upregulation of SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 and TWIST (Katoh, M., Nakagama, H., (2014) "FGF receptors: cancer biology and therapeutics," Med. Res. Rev. 34(2): 280-300). EMT is a cellular process similar to endothelial-to-mesenchymal transition (EndMT). Hypoxia induces angiogenesis as a result of VEGF upregulation (Dewhirst M W, Cao Y, Moeller B. (2008) "Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response." Nat Rev Cancer. 8:425-437). Angiogenesis is orchestrated by the VEGF, FGF, Notch, TGF-β, Hedgehog and WNT signaling cascades, which directly or indirectly regulate the quiescence, migration and proliferation of endothelial cells.
During the earliest stages of angiogenesis, such as in response to the angiogenic cytokine VEGF induced by wounding and ischemia, vascular basement membrane is degraded (Senger, D R, and David, G E, (2011) "Angiogenesis," Cold Spring Harb. Perspect. Biol. Aug. 3(8): a005090 citing Sundberg, C. et al. (2001) "Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/VEGF-164 gene delivery," Am J Pathol 158: 1145-1160; Rowe, R G and Weiss, S J (2008) "Breaching the basement membrane: Who, when and how?" Trends Cell Biol 18: 560-574; Chang S H et al. (2009) "VEGF-A induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules, causing basement membrane degradation and mother vessel formation," Cancer Res 69: 4537-4544). Following disruption of basement membrane, and with the ensuing stage known as vascular sprouting (Id. Citing Nicosia, R F and Madri, J A (1987) "The microvascular extracellular matrix. Developmental changes during angiogenesis in the aortic ring-plasma clot model." Am J Pathol 128: 78-90)), vessels become leaky and hyperpermeable to blood plasma proteins (Id. Citing Sundberg, C. et al. (2001) "Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery." Am J Pathol 158: 1145-1160). This vascular hyperpermeability causes leakage of the plasma proteins fibrinogen, vitronectin, and fibronectin from the blood (Id. Citing Senger, D R (1996) "Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin." Biochim Biophys Acta 1314: 13-24; Sundberg, C. et al. (2001) "Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery." Am J Pathol 158: 1145-1160). Fibrinogen is subsequently converted to fibrin through enzymatic coagulation, and together with extravasated vitronectin and fibronectin instantly transform the interstitial collagen matrix to form a new, provisional ECM. Thus, the early stages of sprouting angiogenesis are generally believed to proceed in an environment rich in preexisting interstitial collagens in combination with fibrin, vitronectin, and fibronectin derived from the blood plasma. As vascular morphogenesis proceeds and vascular sprouts acquire lumens and mature, neovessels are again enshrouded in vascular basement membrane with associated pericytes and thereby achieve stability (Id. Citing Grant, D S and Kleinman, H K (1997) "Regulation of capillary formation by laminin and other components of the extracellular matrix." EXS 79: 317-333; Benjamin, L E et al. (1999) "Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal." J Clin Invest 103: 159-165). Pericyte recruitment to vascular tubes directly controls this basement membrane assembly step in vitro and in vivo (Id. Citing Stratman, A N et al. (2009) "Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation." Blood 114: 5091-5101; Stratman, A N et al. (2010) "Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization." Blood 116: 4720-4730). Thus, in response to stimulation with angiogenic cytokines, angiogenesis in the adult is generally believed to proceed through the following basic stages: (1) degradation of vascular basement membrane and activation of quiescent endothelial cells (ECs); (2) sprouting and proliferation of ECs within provisional ECM; (3) lumen formation within the vascular sprouts, thereby creating vascular tubes; and (4) coverage of vascular tubes with mature vascular basement membrane in association with supporting pericytes.
Neovascularization
While often considered synonymous with angiogenesis (formation of new vessels from existing vessels), neovascularization involves a much broader series of temporally controlled vascular processes beginning with angiogenesis and progressing through multiple phases resulting in the formation of a new functional circulatory network (LeBlanc, A J et al, (2012) "Microvascular repair—post-angiogenesis vascular dynamics," Microcirculation 19(8): 10.1111/j.1549-8719.2012.00207.x). At the onset of neovascularization, relevant microvessel segments relax their stable vessel structure and initiate vessel sprouting leading to the formation of new vessel segments. (Id). Subsequently, the newly formed neovessels remodel via vascular cell differentiation and incorporation of perivascular cells into the newly formed vessel walls resulting in the appropriate density and distribution of arterioles, venules, and capillaries. (Id). Finally, the newly formed vascular network matures and remodels into a more efficient perfusion circuit that meets tissue perfusion needs and function. (Id).
Effective adult tissue neovascularization, whether by native or therapeutic means, results in an expanded vascular network and increased blood perfusion pathway length resulting in the appropriate delivery of more blood to tissues (Id).
While there is no one stereotypical vascular architecture, microvascular networks generally involve a branched network of progressively smaller caliber small arteries/arterioles at the inflow side delivering blood to the distal capillaries which subsequently drain into a branched network of increasingly larger caliber outflow venules/small veins, although there are variations of this basic network organization, often reflecting tissue and/or organ specific function (Id). Each of the three general vascular compartments (arterioles, capillaries, and venules) performs different functions in the microcirculation due to their structural and functional characteristics and their locations within the vasculature (Id). Arterioles provide the greatest resistance to blood flow in the vascular circuit with most of this resistance attributed to 1st and 2nd branch order arterioles (Id. Citing Mayrovitz H N, Wiedeman M P, Noordergraaf A. (1975) "Microvascular hemodynamic variations accompanying microvessel dimensional changes." Microvasc Res. 10:322-29 Box 1). This is primarily due to the relative larger diameter differences between the feeding arteries and the smaller arterioles and the relative fewer numbers of these proximal arterioles. The more prevalent downstream and terminal arterioles act to broadly distribute blood throughout the tissue and control, via vessel tone dynamics, blood flow into the most distal capillaries. The very small diameters and large numbers of capillaries make them ideal for supporting effective blood-tissue exchange of oxygen and other blood nutrients and molecules. Finally, venules, due in part to a relatively more compliant wall, serve as a high capacitance drainage system. Importantly, in a competent microcirculatory bed, as vessel diameters reduce within a vascular compartment the number of vessels in that compartment increase due to branching. This results in a sufficiently large enough cross-sectional area to keep resistance to blood flow across the compartment relatively low even though resistance within a single vessel segment might be high (due to the inverse relationship between resistance and the 4th power of the radius). Thus, to maintain proper resistances across the microvasculature, and therefore effective perfusion, proper branch ordering is critical. In addition, blood flow distribution in a tissue depends on the extent of branching in a logarithmic fashion (Mayrovitz H N, Tuma R F, Wiedeman M P. (1977) "Relationship between microvascular blood velocity and pressure distribution." Am J Physiol.; 232:H400-5). This normalized relationship between vessel caliber and vessel numbers (i.e. branching) is a critical feature of functional microvascular network architectures. Mismatches in this relationship lead to poor hemodynamic function typically observed as hypo-perfusion and/or hypoxia within the tissue. Deficits in blood perfusion (e.g. ischemia, hypoxia) are a cause of and/or complication associated with a number of disease states including tissue infarction, necrosis, wound healing, tissue grafting, and organ dysfunction.
Microvascular Stability: Rho GTPase Cdc42 has been Implicated in the Mediation of Endothelial Barrier Function
Endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind β-catenin through their cytoplasmic domain (Broman, M T et al (2006) "Cdc42 regulates adherens junction stability and endothelial permeability by inducing alpha-catenin interaction with the vascular endothelial cadherin complex," Cir. Res. 98: 73-80). β-catenin in turn binds α-catenin and connects the AJ complex with the actin cytoskeleton (Id). Rho GTPase Cdc42 regulates AJ permeability by controlling the binding of α-catenin with β-catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton (Id). β-catenin and the associated α-catenin may then serve as support sites for actin polymerization, leading to formation of long endothelial plasma membrane protrusions (Kouklis, P. et al (2003) VE-cadherin-induced Cdc42 signaling regulates formation of membrane protrusions in endothelial cells," J. Biol. Chem. 278: 16230-36). Non-junctional VE-cadherin thus actively participates in inside-out signaling at the plasma membrane, leading to the development of endothelial membrane protrusions (Id).
During inflammation, inflammatory mediators increase vascular permeability primarily by formation of intercellular gaps between endothelial cells of post-capillary venules. Spindler, V. et al (2010) "Role of GTPases in control of microvascular permeability," Cardiovasc. Res. 87(2): 243-53). Adherens junctions of endothelial cells need to be dynamic when endothelial junctions transiently open to allow passage of leukocytes from the blood into tissues. Rac1 and Cdc42 are the main GTPases required for barrier maintenance and stabilization. RhoA negatively regulates barrier properties (i.e., renders the barrier more permeable) under both resting and inflammatory conditions (Id). Rho GTPases (RhoA, Rac1 and Cdc42) or Rap1 are known to regulate cell adhesion in part by reorganization of the junction-associated cortical actin cytoskeleton (Id). Activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability (Ramchandran, R. et al (2008) "Critical role of Cdc42 in mediating endothelial barrier protection in vivo," Am. J. Physiol. Lung Cell Mol. Physiol. 295: 363-69), while Rac1-mediated barrier destabilization in microvascular endothelium appears to be largely restricted to conditions of enhanced endothelial cell migration and thus to be more closely related to angiogenesis rather than to inflammation. (Spindler, V. et al (2010) "Role of GTPases in control of microvascular permeability," Cardiovasc. Res. 87(2): 243-53)). Recent studies revealed that cAMP signaling, which is well known to be barrier protective, enhances barrier functions in part via Rap1-mediated activation of Rac1 and Cdc42 as well as by inhibition of RhoA. Moreover, barrier-stabilizing mediators directly activate Rac1 and Cdc42 or increase cAMP levels (Id). On the other hand, several barrier-disruptive components appear to increase permeability by reduced formation of cAMP, leading to both inactivation of Rac1 and activation of RhoA (Id).
The Cholesterol Biosynthesis Pathway
The mevalonate arm of the cholesterol biosynthesis pathway, which includes enzymatic activity in the mitochondria, peroxisome, cytoplasm and endoplasmic reticulum, starts with the consumption of acetyl-CoA, which occurs in parallel in 3 cell compartments (the mitochondria, cytoplasm, and peroxisome) and terminates with the production of squalene in the endoplasmic reticulum (Mazein, A. et al. (2013) "A comprehensive machine-readable view of the mammalian cholesterol biosynthesis pathway," Biochemical Pharmacol. 86: 56-66). The following are enzymes of the mevalonate arm:
Acetyl-CoA acetyltransferase (ACAT1; ACAT2; acetoacetyl-CoA thiolase; EC 22.214.171.124) catalyzes the reversible condensation of two molecules of acetylcoA and forms acetoacetyl-CoA (Id).
Hydroxymethylglutaryl-CoA synthase (HMGCS1 (cytoplasmic); HMGCS2 (mitochondria and peroxisome); EC 18.104.22.168 catalyzes the formation of 3-hydroxy-3-methylglutaryl CoA (3HMG-CoA) from acetyl CoA and acetoacetyl Co A (Id).
Hydroxymethylglutaryl-CoA lysase (mitochondrial, HMGCL; EC 184.108.40.206) transforms HMG-CoA into Acetyl-CoA and acetoacetate.
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR; EC 1.1.34) catalyzes the conversion of 3HMG-CoA into mevalonic acid. This step is the committed step in cholesterol formation. HMGCR is highly regulated by signaling pathways, including the SREBP pathway (Id).
Mevalonate kinase (MVK; ATP: mevalonate 5-phosphotransferase; EC 184.108.40.206) catalyzes conversion of mevalonate into phosphomevalonate (Id).
Phosphomevalonate kinase (PMVK; EC 18.104.22.168) catalyzes formation of mevalonate 5-diphosphate from mevalonate 5-phosphate (Id).
Diphosphomevalonate decarboxylase (MVD; mevalonate (diphospho) decarboxylase; EC 184.108.40.206) decarboxylates mevalonate 5-diphosphate, forming isopentenyldiphosphate while hydrolyzing ATP (Id).
Isopentenyl-diphosphate delta-isomerases (ID11; ID12; EC 184.108.40.206) isomerize isopentenyl diphosphate into dimethylallyl diphosphate, the fundamental building blocks of isoprenoids (Id).
Farnesyl diphosphate synthase (FDPS; EC184.108.40.206; EC 18.104.22.168; dimethylallyltranstransferase) catalyzes two reactions that lead to farnesyl diphosphate formation. In the first (EC 126.96.36.199 activity), isopentyl diphosphate and dimethylallyl diphosphate are condensed to form geranyl disphosphate. Next, geranyl diphosphate and isopentenyl diphosphate are condensed to form farnesyl diphosphate (EC 220.127.116.11 activity) (Id).
Geranylgeranyl pyrophosphate synthase (GGPS1; EC 126.96.36.199; EC 184.108.40.206; farnesyl diphosphate synthase; EC 220.127.116.11; dimethylallyltranstransferase) catalyzes the two reactions of farnesyl diphosphate formation and the addition of three molecules of isopentenyl diphosphate to dimethylallyl diphosphate to form geranylgeranyl diphosphate (Id).
Farnesyl-diphosphate farnesyltransferase 1 (FDFT1; EC 220.127.116.11; squalene synthase) catalyzes a two-step reductive dimerization of two farnesyl diphosphate molecules (C15) to form squalene (C30). The FDFT1 expression level is regulated by cholesterol status; the human FDFT1 gene has a complex promoter with multiple binding sites for SREBP-1a and SREBP-2 (Id).
The sterol arms of the pathway start with Squalene and terminate with cholesterol production on the Bloch and Kandutsch-Russell pathways and with 24 (S),25-epoxycholesterol on the shunt pathway (Id). The following are enzymes of the sterol arms:
Squalene epoxidase (SQLE; EC 18.104.22.168, squalene monooxygenase) catalyzes the conversion of squalene into squalene-2,3-epoxide and the conversion of squalene-2,3-epoxide (2,3-oxidosqualene) into 2,3:22,23-diepoxysqualene (2,3:22,23-dioxidosqualene). The first reaction is the first oxygenation step in the cholesterol biosynthesis pathway. The second is the first step in 24(S),25-epoxycholesterol formation from squalene 2,3-epoxide (Id).
Lanosterol synthase (LSS; OLC; OSC; 2,3-oxidosqualene:lanosterol cyclase; EC 18.104.22.168) catalyzes cyclization of squalene-2,3-epoxide to lanosterol and 2,3:22,23-depoxysqualene to 24(S),25-epoxylanosterol (Id).
Δ(24)-sterol reductase (DHCR24; 24-dehydrocholesterol reductase; EC 22.214.171.124) catalyzes the reduction of the Δ-24 double bond of intermediate metabolites. In particular it converts lanosterol into 24, 25-dihydrolanosterol, the initial metabolite of the Kandutsch-Russel pathway and also provides the last step of the Bloch pathway converting desmosterol into cholesterol. Intermediates of the Bloch pathway are converted by DHCR24 into intermediates of the Kandutsch-Russell pathway (Id).
Lanosterol 14-α demethylase (CYP51A1; cytochrome P450, family 51, subfamily A, polypeptide 1; EC 188.8.131.52) converts lanosterol into 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol and 24,25-dihydrolanosterol into 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in three steps (Id).
Delta (14)-sterol reductase (TM7F2; transmembrane 7 superfamily member 2, EC 220.127.116.11) catalyzes reactions on the three branches of the cholesterol and 24(S),25-epoxycholesterol pathways (Id).
Methylsterol monooxygenase 1 (MSM01; SC4MOL; C-4 methylsterol oxidase; EC 184.108.40.206) catalyzes demethylation of C4 methylsterols (Id).
Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (NSDHL; NAD(P) dependent steroid dehydrogenase-like; EC 126.96.36.199) participates in several steps of post-squalene cholesterol and 24(S),25-epoxycholeseterol synthesis (Id).
3-keto-steroid reductase (HSD17B7; 17-beta-hydroxysteroid dehydrogenase 7; EC 126.96.36.1990) converts zymosterone into zymosterol in the Bloch pathway (Id).
3-β-hydroxysteroid-Δ(8), Δ(7)-isomerase (EBP; emopamil-binding protein; EC126.96.36.199) catalyzes the conversion of Δ(8)-sterols into Δ(7)-sterols (Id).
Lathosterol oxidase (SC5DL; sterol-C5-desaturase (ERG3 Δ-5-desaturase homolog, S. cerevisiae-like; EC 220.127.116.11) catalyzes the production of 7-dehydrocholesterol, 7-dehydrodesmosterol and 24(S),25-epoxy-7-dehydrocholesterol (Id).
7-dehydrocholesterol reductase (DHCR7; EC 126.96.36.199) catalyzes reduction of the C7-C8 double bond of 7-dehydrocholesterol and formation of cholesterol, and produces desmosterol from 7-dehydrodesmosterol and 24(S),25-epoxycholesterol from 24(S),25-epoxy-7-dehydrocholesterol (Id).
Cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4; 1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6α-hydroxylase; EC 126.96.36.199)) catalyzes the hydroxylation of cholesterol leading to 25-hydroxycholesterol and 4β-hydroxycholesterol (Id).
Cholesterol 25-hydroxylase (CH25H; cholesterol 25-monooxygenase; EC 126.96.36.199) uses di-iron cofactors to catalyze the hydroxylation of cholesterol to produce 25-hydroxycholesterol, and has the capacity to catalyze the transition of 24-hydroxycholesterol to 24, 25-dihydroxycholesterol (Id).
Cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1; cholesterol 7-alpha-hydroxylase; EC 126.96.36.199) is responsible for introducing a hydrophilic moiety at position 7 of cholesterol to form 7α-hydroxycholesterol (Id).
Cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1; Sterol 27-hydroxylase; EC 126.96.36.199) catalyzes the transition of mitochondrial cholesterol to 27-hydroxycholesterol and 25-hydroxycholesterol (Id).
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase, EC 220.127.116.11) catalyzes transformation of cholesterol into 24(S)-hydroxycholesterol (Id).
Statins
The term "statin" as used herein refers to a competitive inhibitor of HMG-CoA reductase in the mevalonate arm of the cholesterol biosynthesis pathway. Exemplary statins include, without limitation, mevastatin, lovastatin, simvastatin, and pravastatin, which are fungal metabolites, and fluvastatin, atorvastatin, and verivastatin, which are synthetic compounds. Statins exert their major effect—reduction of low density lipoprotein cholesterol levels—through a mevalonic acid-like moiety that competitively inhibits HMGCR by product inhibition. Higher doses of the more potent statins (e.g., atorvastatin and simvastatin) also can reduce triglyceride levels caused by elevated very low density lipoprotein levels (Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ed. Joel G. Hardman, Lee E. Limbird, Eds., 10th Ed., McGraw Hill, New York (2001), p. 984).
HMG-CoA reductase inhibition by the statins cerivastatin and atorvastatin has been shown to have a biphasic dose-dependent effect on angiogenesis that is lipid independent and associated with alterations in endothelial apoptosis and VEGF signaling. (Weis, M. et al (2002) "Statins have biphasic effects on angiogenesis," Cir. Res. 105: 739-45). Endothelial cell proliferation, migration, and differentiation of an immortalized human dermal microvascular endothelial cell line (HMEC-1) in vitro were enhanced at low statin concentrations (0.005 to 0.01 μmol/L) but significantly inhibited at high statin concentrations (0.05 to 1 μmol/L). Antiangiogenic effects at high concentrations were associated with decreased endothelial release of VEGF and increased endothelial apoptosis and were reversed by geranylgeranyl pyrophosphate (GGP). GGP is required for the membrane localization of Rho family members. Other antiangiogenic effects of statins may include inhibition of the expression or activity of monocyte chemoattractant protein-1, metalloproteinase and angiotensin-2, preproendothelin gene, and actin filament and focal adhesion formation. In a zebrafish anti-angiogenic drug screen, a number of statins (simvastatin, mevastatin, lovastatin, and rosuvastatin) were identified to inhibit angiogenesis in developing zebrafish embryos. The anti-angiogenic effect of rosuvastatin was confirmed in a mouse xenograft prostate cancer model. (Wang, C. et al, (2010) "Rossuvastatin, identified from a zebrafish chemical genetic screen for anti-angiogenic compounds, suppresses the growth of prostate cancer," Eur. Urol. 58: 418-26). In other murine models, inflammation-induced angiogenesis was enhanced with low-dose statin therapy (0.5 mg/kg/d) but significantly inhibited with high concentrations of cerivastatin or atorvastatin (2.5 mg/kg/d). Despite the fact that high-dose statin treatment was effective at reducing lipid levels in hyperlipidemic apolipoprotein E-deficient mice, it impaired, rather than enhanced angiogenesis.
Prenylation
Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins (Zhang, F. L. and Casey, P J (1996) "Protein Prenylation: Molecular Mechanisms and Functional Consequences," Ann. Rev. Biochem. 65: 241-69). Prenylation promotes membrane interactions of prenylated proteins, and plays a major role in several protein-protein interactions involving them.
Both the 15-carbon isoprenoid FPP and the 20-carbon isoprenoid GGP are products of the MVA metabolic pathway; it follows that regulation of HMGCR, FTase and GGTase-I, the key enzymes of the mevalonate pathway, can significantly affect the protein prenylation process. Zhang, F. L. and Casey, P J (1996) "Protein Prenylation: Molecular Mechanisms and Functional Consequences," Ann. Rev. Biochem. 65: 241-69).
Prenylated proteins can be grouped into two major classes: those containing the CAAX motif and the so-called CC- or CxC-containing proteins. CAAX proteins are defined as a group of proteins with a specific amino acid sequence at C-terminal that directs their post translational modification. Gao, J. et al (2009) "CAAX-box protein, prenylation process and carcinogenesis," Am. J. Trans. Res. 1(3): 312-25). C is cysteine residue, AA are two aliphatic residues, and X represents any C-terminal amino acid depending on different substrate specificity. The CAAX proteins encompass a wide variety of molecules that include nuclear lamins (intermediate filaments), Ras and a multitude of GTP-binding proteins (G proteins), and several protein kinases and phosphatases. Most CAAX proteins are found primarily at the cytoplasmic surface of cell membranes and are involved in a tremendous number of cellular signaling processes and regulatory events that play various roles in cell biological functions. These activities include cell proliferation, differentiation, nuclear stability, embryogenesis, spermatogenesis, metabolism, and apoptosis. The proteins that have a CAAX box at the end of the C-terminal always need a prenylation process before the proteins can be sent to plasma membrane or nuclear membrane and thereby exert their different functions.
Prenylation of CAAX proteins includes 3 steps: polyisoprenylation, proteolysis, and carboxyl methylation. Zhang, F. L. and Casey, P J (1996) "Protein Prenylation: Molecular Mechanisms and Functional Consequences," Ann. Rev. Biochem. 65: 241-69). First, an isoprenoid lipid is attached to the CAAX box by a prenyltransferase, for example, FTase or GGTase-I. When the C terminal amino acid "X" is serine, methionine or glutamine, proteins are recognized by FTase, whereas a leucine at this position results in modification by GGTase I. FTase and GGTase-I recognize the CAAX box in the protein, and then add the 15-carbon isoprenoid farnesyl pyrophosphate by FTase or the 20-carbon isoprenoid by GGTase-I to the cysteine residue of the CAAX box. Second, following prenylation, the aaX residues are cleaved by an endoprotease. Third, the carboxyl group of the modified cysteine is methylated by a specific methyl transferase.
GGTase II transfers geranylgeranyl groups from GGPP to both cysteine residues of CC- or CxC-containing proteins in a process mechanistically distinct from that of CAAX proteins. Additionally, proteins containing the CxC motif are methylated at the C-terminal prenylcysteine, whereas CC-containing proteins are not.
HMGCR mediated GGPP biosynthesis regulates Cdc42 prenylation. (Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W (2013) "The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signaling pathway," Dev Biol 373:258-266). Cdc42 regulates adherens junction stability and endothelial barrier function.
Intracerebral Hemorrhage (ICH)
Spontaneous intracerebral hemorrhage (ICH) is a severe and debilitating form of stroke that is most commonly due to hypertension, amyloid angiopathy, brain vascular malformations or secondary to medications including antiplatelet and anticoagulant drugs. Spontaneous ICH comprises 10% of strokes and is associated with death or disability in more than 50% of the approximately 90,000 patients affected each year in North America. (Roger V L, Go A S, Lloyd-Jones D M, Adams R J, Berry J D, Brown T M, et al (2011) "Heart disease and stroke statistics—2011 update: a report from the American Heart Association," Circulation 123:e18-e209). Clinical studies also have disclosed a link between cholesterol-lowering HMGCR inhibitors (statins) and increased risk of ICH. (Collins R, Armitage J, Parish S, Sleight P, Peto R: (2004) "Effects of cholesterol-lowering with simvastatin on stroke and other major vascular events in 20536 people with cerebrovascular disease or other high-risk conditions," Lancet 363:757-767); Flaster M, Morales-Vidal S, Schneck M J, Biller J (2011) "Statins in hemorrhagic stroke," Expert Rev Neurother 11:1141-1149; Goldstein L B, Amarenco P, Szarek M, Callahan A, III, Hennerici M, Sillesen H, et al (2008) "Hemorrhagic stroke in the Stroke Prevention by Aggressive Reduction in Cholesterol Levels study," Neurology 70:2364-2370). Another type of brain hemorrhage is brain microhemorrhages (BMH), which are small, usually multiple, ICHs. A systematic review found that 5% of healthy adults, 34% of patients with ischemic stroke and 60% of patients with nontraumatic ICH had BMH. (Cordonnier C, Klijn C J, van B J, Al-Shahi S R (2010) "Radiological investigation of spontaneous intracerebral hemorrhage: systematic review and trinational survey," Stroke 41:685-690). They are more common in patients with hypertension and diabetes mellitus. Other than treatment of hypertension, there is no prophylactic treatment to prevent ICH or BMH.
1. Intracerebral Hemorrhage, Brain Microhemorrhages and other Causes of Intracerebral Hemorrhage
Spontaneous ICH accounts for 10% of strokes. There are about 90,000 per year in the U.S. and Canada. Mortality is 30-50%. The most common cause is hypertension, and ICH due to hypertension can be partly reduced by treating hypertension. However, other factors contribute to ICH from hypertension, such as low serum cholesterol (Sutherland G R, Auer R N (2006) "Primary intracerebral hemorrhage," J Clin Neurosci 13:511-517). The second main cause of ICH is amyloid angiopathy, for which there is no specific treatment. The pathophysiology of ICH from amyloid blood vessels is unknown, although it is highly associated with amyloid deposition in brain arteries and arterioles.
Brain microhemorrhages (BMH) are another form of ICH (Fisher M J (2013) "Brain regulation of thrombosis and hemostasis: from theory to practice," Stroke 44:3275-3285). They are associated with increasing age, amyloid angiopathy, hypertension, ischemic/hemorrhagic stroke (mixed cerebrovascular disease) and Alzheimer disease. They are usually attributed to localized bleeding from tears in small arterioles but Fisher proposed that they may be age-dependent, inflammation-mediated leakage from small brain blood vessels (Id). This hypothesis is supported by BMH induced by lipopolysaccharide (LPS) in zebrafish and mice (FIGS. 5, 6 and 9) (Liu S, Vasilevko V, Cribbs D H, Fisher M (2013) "A mouse model of cerebral microhemorrhages," Stroke 44:AWP297 (Abstract)). Furthermore, patients with BMH are at increased risk of ICH and that risk is increased further if they take antiplatelet or anticoagulant drugs (Cordonnier C, Klijn C J, van B J, Al-Shahi S R (2010) "Radiological investigation of spontaneous intracerebral hemorrhage: systematic review and trinational survey," Stroke 41:685-690; Greenberg S M, Eng J A, Ning M, Smith E E, Rosand J (2004) "Hemorrhage burden predicts recurrent intracerebral hemorrhage after lobar hemorrhage," Stroke 35:1415-1420; Imaizumi T, Horita Y, Hashimoto Y, Niwa J (2004) "Dotlike hemosiderin spots on T2*—Weighted magnetic resonance imaging as a predictor of stroke recurrence: a prospective study," J Neurosurg 101:915-920). BMHs also are associated with cognitive impairment (Yakushiji Y, Noguchi T, Hara M, Nishihara M, Eriguchi M, Nanri Y, et al (2012) "Distributional impact of brain microhemorrhages on global cognitive function in adults without neurological disorder," Stroke 43:1800-1805).
While statins reduce the long-term risk of myocardial infarction and ischemic stroke, they increase the risk of ICH (Collins R, Armitage J, Parish S, Sleight P, Peto R (2004) "Effects of cholesterol-lowering with simvastatin on stroke and other major vascular events in 20536 people with cerebrovascular disease or other high-risk conditions," Lancet 363:757-767; Flaster M, Morales-Vidal S, Schneck M J, Biller J (2011) "Statins in hemorrhagic stroke," Expert Rev Neurother 11:1141-1149; Goldstein L B, Amarenco P, Szarek M, Callahan A, III, Hennerici M, Sillesen H, et al (2008) "Hemorrhagic stroke in the Stroke Prevention by Aggressive Reduction in Cholesterol Levels study," Neurology 70:2364-2370, Haussen D C, Henninger N, Kumar S, Selim M (2012) "Statin use and microhemorrhages in patients with spontaneous intracerebral hemorrhage," Stroke 43:2677-2681); Eisa-Beygi S, Wen X Y, Macdonald R L. (2014) "A Call for Rigorous Study of Statins in Resolution of Cerebral Cavernous Malformation Pathology." Stroke 45(6):1859-61. According to the American Heart Association guidelines, statins may not be indicated in these patients. (Morgenstem L B, Hemphill J C, III, Anderson C, Becker K, Broderick J P, Connolly E S, Jr., et al (2010) "Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/American Stroke Association," Stroke 41:2108-2129). Statins inhibit cholesterol synthesis, and low serum cholesterol also is an independent risk factor for ICH. (Sutherland G R, Auer R N (2006) "Primary intracerebral hemorrhage," J Clin Neurosci 13:511-517).
Cerebral cavernous malformations (CCM) are the most common brain vascular malformation. They are found in 0.5% of the population and are a cause of spontaneous ICH (Richardson B T, Dibble C F, Borikova A L, Johnson G L (2013) "Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling," Biol Chem 394:35-42). The hemorrhages tend to cluster in time so a drug that reduced this risk during times of increased hemorrhage risk is actively being sought and is greatly needed (Barker F G, Amin-Hanjani S, Butler W E, Lyons S, Ojemann R G, Chapman P H, et al (2001) "Temporal clustering of hemorrhages from untreated cavernous malformations of the central nervous system," Neurosurgery 49:15-24, Li Q, Mattingly R R (2008) "Restoration of E-cadherin cell-cell junctions requires both expression of E-cadherin and suppression of ERK MAP kinase activation in Ras-transformed breast epithelial cells," Neoplasia 10:1444-1458). CCM may be sporadic or inherited in association with loss-of-function mutations in genes encoding 3 structurally distinct proteins, CCM1 (KRIT1), CCM2 (Osmosensing scaffold for MEKK3 or OSM, MALCAVERIN, or MGC4607), and CCM3 (programmed cell death 10 (PDCD10)(Li D Y, Whitehead K J (2010) "Evaluating strategies for the treatment of cerebral cavernous malformations," Stroke 41:S92-S94). All 3 CCM proteins are involved in cytoskeleton and AJ and the mutations have to be in endothelial cells in order for CCMs to form. Mutations in CCM1 and CCM2 lead to increased RhoA activity, which led to the hypothesis that increased RhoA activity affects the cell cytoskeleton and causes vascular instability, CCMs and possibly ICH/BMH in humans. Drugs that inhibit RhoA activity, such as statins and fasudil, are theorized to reduce the risk of ICH based on data from mouse models of CCMs. (Li D Y, Whitehead K J (2010) "Evaluating strategies for the treatment of cerebral cavernous malformations," Stroke 41:S92-S94; Richardson B T, Dibble C F, Borikova A L, Johnson G L (2013) "Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling," Biol Chem 394:35-42). This hypothesis is in contrast to studies in zebrafish showing that statins impair vascular stability and give rise to ICH/BMH (Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W (2013) "The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signaling pathway," Dev Biol 373:258-266). These effects were shown to be due to defective prenylation of Rho GTPases, particularly Cdc42, a Rho GTPase involved in the regulation of vascular stability, leading to the question as to the cause of the discrepancy between zebrafish and mouse models. In fact, statin treatment of zebrafish induces cerebrovascular defects typified by leaky, dilated cranial vessels with sluggish blood flow, which are analogous to CCMs. Without being limited by theory, it is hypothesized herein that the difference is due to relative degrees of inhibition of RhoA and Cdc42, since the balance of vascular destabilizing RhoA to vascular stabilizing Cdc42 may differ depending on dose and species.
2. Models of ICH and BMH
Zebrafish are emerging as useful model organism for large-scale, phenotype-based chemical and genetic screening. Zebrafish are genetically very similar to humans, easy and fast to breed for high-throughput screening, transparent early on for easy imaging and relatively easy to modify genetically. Some compounds discovered in zebrafish are effective in mammals and already in human studies (Peterson R T, Fishman M C (2011) "Designing zebrafish chemical screens," Methods Cell Biol 105:525-541). Since the screening is performed in vivo, general drug toxicity can be evaluated at the same time as drug efficacy, allowing for a higher success rate as compared to an in vitro drug screens on cultured cells (Miscevic F, Rotstein O, Wen X Y (2012) "Advances in zebrafish high content and high throughput technologies," Comb Chem High Throughput Screen 15:515-521, 2012). Furthermore, an in vivo screen system such as zebrafish can measure the efficacy of the drug as well as its metabolites. The ability to perform high throughput screening of compound libraries in zebrafish is an advantage over testing compounds in rodents where high throughput screening is not possible.
Several models of ICH/BMH have been identified in zebrafish. First, statins cause ICH/BMH in zebrafish (FIG. 7, 9) (Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W (2013) "The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signaling pathway," Dev Biol 373:258-266). The mechanism is due to inhibition of protein prenylation since ICH/BMH can also be induced by MO-induced depletion of the β subunit of geranylgeranyltransferase 1 (GGTase 1, pggtlβ) and prevented by downstream metabolic rescue with the product of HMGCR, geranylgeranyl pyrophosphate (GGPP, FIGS. 3, 4 and 8). GGPP is a 20 carbon lipid molecule required for post-translational prenylation of Rho GTPase proteins. The BMH temporal and spatial distribution is similar to ICH/BMH seen in zebrafish bubblehead (bbhm292) and redhead (rhdmi149) mutants. (Buchner D A, Su F, Yamaoka J S, Kamei M, Shavit J A, Barthel L K, et al (2007) "pak2a mutations cause cerebral hemorrhage in redhead zebrafish," Proc Natl Acad Sci USA 104:13996-14001; Butler M G, Gore A V, Weinstein B M (2011) "Zebrafish as a model for hemorrhagic stroke," Methods Cell Biol 105:137-161; Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al (2007) "A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish," Proc Natl Acad Sci USA 104:13990-13995).
Bubblehead (bbhm292) develops ICH and brain edema 36 to 52 hours postfertilization (hpf) whereas redhead (rhdmi149) develops ICH 2 to 3 days postfertilization. The bbhm292 mutation is in the βpix (pak-interacting exchange factor β) gene, whereas the rhdmi149 mutation is in pak2a (p21 protein [Cdc42/Rac]-activated kinase 2a) gene. These genes encode proteins that regulate activity of Rho GTPases, Rac and Cdc42. That both of these changes are associated with ICH/BMH is consistent with Rac and Cdc42 requiring GGTase 1-mediated prenylation. GGTase 1 post-translationally modifies Rac and Cdc42 by adding a mevalonate-derived GGPP which is required to activate these GTPases. (Peterson Y K, Kelly P, Weinbaum C A, Casey P J (2006) "A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity," J Biol Chem 281:12445-12450) There are also zebrafish mutants corresponding to the orthologous human CCM1, CCM2 and CCM3 genes (Butler M G, Gore A V, Weinstein B M (2011) "Zebrafish as a model for hemorrhagic stroke," Methods Cell Biol 105:137-161). These develop cardiac dilation and progressively enlarged, dilated blood vessels and it has been suggested the genes have similar function in both species. No ICH phenotype is described in these zebrafish mutants but combined MO-induced reduction in a Ras GTPase effector protein, rap 1b and zebrafish ccm1 did cause ICH (Gore A V, Lampugnani M G, Dye L, Dejana E, Weinstein B M (2008) "Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke," Dis Model Mech 1:275-281). In mice, the gene defects for CCMs are suggested to be required in endothelial cells in order for malformations to develop (Chan A C, Li D Y, Berg M J, Whitehead K J (2010) "Recent insights into cerebral cavernous malformations: animal models of CCM and the human phenotype," FEBS J 277:1076-1083).
Mouse models of ICH include direct injection of blood into the brain, or injection of elastase, which degrades vascular collagen and causes bleeding. These models would not be useful for detecting therapeutic agents that stabilize the vasculature. There are two models of spontaneous ICH in mice. One is a model of acute and chronic hypertension induced by a combination of angiotensin 2 and NOS inhibition in mice (Wakisaka Y, Chu Y, Miller J D, Rosenberg G A, Heistad D D (2010) "Spontaneous intracerebral hemorrhage during acute and chronic hypertension in mice,". J Cereb Blood Flow Metab 30:56-69). The mechanism of ICH in hypertension, however, may differ from what we are investigating with statins and CCM genes.
A second model of BMH involves transgenic mice (e.g. Tg2576) that spontaneously overexpress P-amyloid, mimicking cerebral amyloid angiopathy (Herzig M C, Winkler D T, Burgermeister P, Pfeifer M, Kohler E, Schmidt S D, et al. Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis. Nat Neurosci. 2004; 7(9):954-60., Fisher M, Vasilevko V, Passos G F, Ventura C, Quiring D, Cribbs D H. Therapeutic modulation of cerebral microhemorrhage in a mouse model of cerebral amyloid angiopathy. Stroke. 2011; 42(11):3300-3). There are other similar models. (Alharbi B M, Tso M K, Macdonald R L. (2016) Animal models of spontaneous intracerebral hemorrhage. Neurol Res 38:448-455). The limitation is that it takes up to 2 years for animals to develop BMH.
LPS has been used to induce BMH in mice (Tang, A T, et al, "Endothelial TLR4 and the microbiome drive cerebral cavernous malformations," Nature (2017) 545 (7654): 305-310. Doi: 10.1038/nature22075; Liu S, Vasilevko V, Cribbs D H, Fisher M (2013) "A mouse model of cerebral," Stroke 44:AWP297 [Abstract]); Liu S, Grigoryan M M, Vasilevko V, Sumbria R K, Paganini-Hill A, Cribbs D H, et al. (2014) "Comparative analysis of H & E and prussian blue staining in a mouse model of cerebral microbleeds." J Histochem Cytochem. 62:767-773). LPS or vehicle (phosphate buffered saline [PBS]) was injected at baseline and again at 24 hours, and the mice were sacrificed 2 days after the first injection (FIGS. 6 and 9). When the brains were examined, multiple small fresh hemorrhages were found in mice treated with LPS, as was increased blood brain barrier permeability. It has been suggested that this model might be useful to study mechanisms of and interventions for BMH.
Anti-β3 Integrin Mouse Model of Intracerebral Hemorrhage (ICH)
The integrin αIIbβ3 is the most abundant glycoprotein on platelets. The β3 subunit also is coexpressed with the αV subunit (i.e., αVβ3) on proliferating endothelial cells (ECs) during angiogenesis (Yougbare, I. et al., "Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage," (2015) J. Clin. Invest. 125(4): 1545-56 citing Brooks, P C et al, "Requirement of vascular integrin alpha v beta 3 for angiogenesis," Science (1994) 264 (5158): 569-71; Brooks, P C et al, "Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels," (1994) Cell 79(7): 1157-64; Di Q, et al, "Impaired cross-activation of β3 integrin and VEGFR-2 on endothelial progenitor cells with aging decrases angiogenesis in response to hypoxia," Intl J. Cardiol. (2013) 168(3): 2167-76; Stupack, D C, Cheresh, D A, "Integrins and angiogenesis," Curr. Top. Dev. Bio. (2004) 64: 207-38. Several studies have demonstrated that β3 plays an important role in angiogenesis. For example, it has been shown that αVb3 was required for angiogenesis (Id. citing Brooks, P C et al, "Requirement of vascular integrin alpha v beta 3 for angiogenesis," Science (1994) 264 (5158): 569-71), and that αVb3 antagonists promoted tumor regression by inducing apoptosis of angiogenic blood vessels (Id. citing Brooks, P C et al, "Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels," (1994) Cell 79(7): 1157-64). Evidence has also shown that integrin αVβ3 cooperated with VEGFR2 in pro-angiogenic signaling (Id., citing Robinson, S D, et al, "αvβ3 integrin limits the contribution of neuropilin-1 to vascular endothelial growth factor-induced angiogenesis," J. Biol. Chem. (2009) 284(49): 33966-81; Soldi, R. et all, "Role of αvβ3 integrin in the activation of vascular endothelial growth factor receptor-2," EMBO J. (1999) 18(4): 882-92) and that AKT phasphorylation was essential in VEGF-mediated post-natal angiogenesis (Id. citing Kitamura, T et al, "Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin," Nat. Cell Biol. (2008): 10(3): 329-337).
An established murine model of fetal and neonatal autoimmune thrombocytopenia (FNAIT) has been used to investigate the mechanism of ICH in affected fetuses and neonates (Id., citing Chen, P. et al., "Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy," Blood (2010) 116 (18): 3660-68; Li, C. et al,"The maternal immune response to fetal platelet GpIbα causes frequent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRn therapies," J. Clin. Invest. (2011) 121(11): 4537-47; Ni H, et al, "A novel murine model of fetal and neonatal alloimmune thrombocytopenia: respons to intravenous IgG therapy," Blood (2006) 107(7): 2976-83). Itgb3−/− and Gp1ba−/− mice (referred to hereinafter as β3−/− and GPIbα−/−) were transfused with WT platelets to mimic exposure to β3 or to GPIbα during conception. Id. Anti-β3 or anti-GPIbα antibodies were detected; these immunized mice were subsequently bred with WT males. Id. Similar severity of thrombocytopenia in the heterozygote (−/+) neonates delivered from immunized β3−/− and GPIBα−/− mice was found. Id. ICH was found in the β3−/− fetuses starting around EC15.5 as well as in neonates using a high-frequency ultrasound imaging system to detect in utero ICH in pregnant mice, and performing H & E staining of brain sections. Id. Hemorrhage was observed in different areas of the brain, and the frequency of ICH increased in fetuses in accordance with the number of material immunizations. Id. ICH was never found in anti-GPIbα-mediated FNAIT fetuses or neonates. Id.
The following experiments showed that anti-β3 antibodies, but not anti-GPIbα antibodies or thrombocytopenia alone, were the cause of ICH. To confirm that ICH was indeed antibody mediated, β3−/− and GPIBα−/− neonates delivered from naïve mice were passively injected with antisera at P2. Postnatal injection of anti-33 sera into β3−/− neonates induced ICH, but anti-GPIbα sera did not induce any ICH in GPIBα−/− neonates (P<0.01). Id. To further determine whether platelet-mediate cytotoxicity (Id. Citing Nieswandt, B. et al., "Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice," Blood (2000) 96(7): 2520-27; Nieswandt, B. et al, "Targeting of platelet integrin αIIbβ3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory FcγR," Intl Immunol. (2003) 15(3): 341-49) might be involved in the mechanism of ICH, anti-β3 sera were injected into αIIb integrin-deficient pups that did not express αIIbβ3 integrin on their platelets. Id. ICH was observed in Itga2b−/− pups with normal platelet counts, and postnatal injection of anti-β3 sera into β3−/− neonates failed to induce ICH and impair retinal vascular development in these antigen-negative pups. Id.
Mouse models that combine Ccm heterozygotes on a background of homozygous deletion of the mismatch repair complex protein Msh2 (<Ccm1+/−Msh−/− and Ccm2+/′Msh−/−) develop CCMs. (McDonald D A, Shi C, Shenkar R, Stockton R A, Liu F, Ginsberg M H, et al, (2012) "Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease," Stroke 43:571-574. Another important gene is Rap1b, mouse mutants of which develop normally until embryonic day 12.5, at which point 50% die due to hemorrhage (Id); Chrzanowska-Wodnicka M (2013) "Distinct functions for Rap1 signaling in vascular morphogenesis and dysfunction," Exp Cell Res 319:2350-2359). Subphenotypic levels of reduction of ccm1 and rap1b in zebrafish cause brain hemorrhage.
3. Mechanisms of ICH and BMH
In patients with hypertension, the cause of ICH is arteriolosclerosis of the small penetrating arteries that tend to arise from large conducting cerebral arteries. (Auer, R N, Sutherland, G R (2005) "Primary intracerebral hemorrhage: pathophysiology," Can. J. Neurol. Sci. 32 Suppl. 2: 3-12). The only currently available treatment is prophylactic treatment of hypertension. Guidelines for management of patients once they have a hypertensive ICH are published, and recommend surgical evacuation of space-occupying cerebellar ICH and general medical supportive care. (Hemphill J C, 3rd, Greenberg S M, Anderson C S, Becker K, Bendok B R, Cushman M, 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).
There are different theories as to why statins increase the risk of ICH. For example, statin-associated ICH and other types of ICH/BMH may be due to defects in the HMGCR pathway (Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W (2013) "The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signaling pathway," Dev Biol 373:258-266). Inhibition of HMGCR or of other downstream molecules such as geranylgeranyltransferase 1 (GGTase 1) causes ICH/BMH in zebrafish embryos (Id). Flaster and colleagues suggested that statins could cause changes in platelets or in the interactions between clotting and fibrinolytic cascades that could promote ICH, although there is no evidence for this thus far (Flaster M, Morales-Vidal S, Schneck M J, Biller J (2011) "Statins in hemorrhagic stroke," Expert Rev Neurother 11:1141-1149).
There are at least 2 mutations that cause ICH in zebrafish. Bubblehead is a loss of function mutation of βpix (p isoform of the p21-activating kinase (Pak)-interacting exchange factor) (Liu J, Zeng L, Kennedy R M, Gruenig N M, Childs S J (2012) "betaPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin alphavbeta8," Dev Biol 363:95-105). βpix regulates vascular stability and βpix mutation is the cause of vascular fragility and ICH in the bbhm292 mutant. (Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al (2007) "A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish," Proc Natl Acad Sci USA 104:13990-13995). βpix also is involved in focal adhesion complexes, which contain integrins and cadherins (Frank S R, Hansen S H (2008) "The PIX-GIT complex: a G protein signaling cassette in control of cell shape," Semin Cell Dev Biol 19:234-244). Another zebrafish mutant, redhead, is rhdmi149 that has a mutation pak2a (p21 protein [Cdc42/Rac]-activated kinase 2a) Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al (2007) "A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish," Proc Natl Acad Sci USA 104:13990-13995). pak2a is a kinase acting downstream of Cdc42 and Rac, and may be involved in a complex with βPix, paxillin and GIT1. Since Cdc42 also is required for interaction of VE-cadherin and the actin cytoskeleton, the defect in the bbhm292 mutant could also be due to defects in this latter interaction. In mice, germ-line or endothelial cell specific Pak2 knockout is embryonic lethal likely due to impaired blood vessel formation. (Radu M, Semenova G, Kosoff R, Chemoff J (2014) "PAK signalling during the development and progression of cancer," Nat Rev Cancer 14:13-25). Both are transmembrane proteins that connect the intracellular cytoskeleton to the extracellular matrix (Frank S R, Hansen S H (2008) "The PIX-GIT complex: a G protein signaling cassette in control of cell shape," Semin Cell Dev Biol 19:234-244; van der Flier A, Sonnenberg A (2001) "Function and interactions of integrins," Cell Tissue Res 305:285-298). They are important in angiogenesis and vascular stability. For example, homozygous integrin αv or β8 null mice die perinatally or before from ICH (Zhu J, Motejlek K, Wang D, Zang K, Schmidt A, Reichardt L F (2002) "β8 integrins are required for vascular morphogenesis in mouse embryos," Development 129:2891-2903). Targeted inactivation of VE-cadherin and truncation of the β-catenin-binding cytosolic domain of VE-cadherin in mice induces endothelial cell-specific apoptosis, in addition to defective remodeling and maturation of the vasculature and early lethality (Carmeliet P, Lampugnani M G, Moons L, Breviario F, Compemolle V, Bono F, et al (1999) "Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis," Cell 98:147-157). Most recently, we demonstrated that by delivering anti-β3 integrin antibody into pregnant mice could induce ICH in mouse embryos and neonatal mice. (Yougbaré, I, Lang S, Yang H, Chen P, Zhao X, Tai W S, Zdravic D, Vadasz B, Li C, Piran S, Marshall A, Zhu G, Tiller H, Killie M K, Boyd S, Leong-Poi H, Wen X Y, Skogen B, Adamson S L, Freedman J and Ni H (2015) "Maternal anti-platelet β3 integrin antibodies impair angiogenesis and cause intracranial hemorrhage in fetal and neonatal alloimmune thrombocytopenia," J. Clinical Investigation (JCI), 125:1545-56). Morpholino-induced reduction of cdh5, the zebrafish homologue of the VE-cadherin gene in humans, causes vascular instability, defective lumen formation and ICH by 48 hpf (Montero-Balaguer M, Swirsding K, Orsenigo F, Cotelli F, Mione M, Dejana E (2009) "Stable vascular connections and remodeling require full expression of VE-cadherin in zebrafish embryos," PLoS ONE 4:e5772). It is known that integrins are linked to βPix in focal adhesions by proteins including G protein-coupled receptor kinase interacting target (GIT1), which is an Arf GTPase activating protein (GAP) (Liu J, Zeng L, Kennedy R M, Gruenig N M, Childs S J (2012) "betaPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin alphavbeta8," Dev Biol 363:95-105). Mice without GIT1 have increased pulmonary vascular density and pulmonary hemorrhage. In zebrafish GIT1 is initially ubiquitously expressed but it becomes restricted to the head by 48 hpf. Knock down of GIT1 expression with MOs causes increases in ICH. Liu, et al., conducted a series of experiments in zebrafish that suggest that βPix interacts with αv and β8 integrins and GIT 1 to stabilize the cerebral vasculature, and inhibiting expression of any of the components causes ICH (Liu J, Zeng L, Kennedy R M, Gruenig N M, Childs S J (2012) "betaPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin alphavbeta8," Dev Biol 363:95-105).
CCM1, 2 and 3 may form a multiprotein complex that also includes integrin β1-binding protein (ICAP-1), the GTPases Rac and Rap1 and MAPK kinase kinase MEKX3 Chrzanowska-Wodnicka M (2013) "Distinct functions for Rap1 signaling in vascular morphogenesis and dysfunction," Exp Cell Res 319:2350-2359; Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al (2007) "A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish. Proc Natl Acad Sci USA 104:13990-13995). This complex modulates adherens junctions by interacting with β catenin and VE-cadherin mainly in endothelial cells (but probably other perivascular cells) and achieving vascular stability. Effects of knockouts of the ccm genes in zebrafish and mice are not fully explored but show that embryonic germ-line knockouts tend not to resemble the human phenotype, although in some cases, conditional, endothelial cell specific knockouts do. In zebrafish, knockdown of rap1b leads to ICH, but also sub-phenotype levels of reduction in rap1b combined with ccm1 leads to ICH.
It is likely that the cellular localization and other factors influence the effect of GTPase signaling on vascular stability and ICH/BMH because in mice with mutations in germ-line or endothelial cell-specific mutations in CCM2, statin inhibition of HMGCR, which reduces Rho GTPase prenylation, improves endothelial integrity and prevents the increased vascular permeability. In zebrafish, however, inhibition of HMGCR with statins or mutation and MO-induced loss of function of proteins that activate Rho GTPase signaling increase vascular permeability (Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al (2007) "A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish," Proc Natl Acad Sci USA 104:13990-13995). The discrepancy is likely due to cell-specific effects, age of the organisms, species differences, differential effects on RhoA and cdc42 (that tend to have opposing effects, RhoA destabilizing cdc42 stabilizing vasculature) or differences between wild-type and CCM2 animals.
Statement of the Problem
Intracerebral hemorrhage, BMH and cavernous malformations share common elements of vascular instability in blood vessels in the brain that lead to intracranial hemorrhage, to brain injury and to death and disability. Intracerebral hemorrhage is the most lethal and devastating type of stroke. There is no pharmacologic treatment available to reduce this and there is a high unmet medical need. Therefore, a need exists for a pharmaceutical composition comprising a therapeutic amount of a vascular stabilizing agent, in some embodiments formulated as a sustained release preparation, that when administered, is effective to prevent or reduce the incidence ICH, BMH and ICH from various causes including brain cavernous malformations.

Small molecule therapeutic compounds that reduce the incidence of intracerebral hemorrhage and brain microhemorrhages


A clip member for sealing an opening in a sheet material includes a body which is fluid tight in an axial direction, at least one flange portion adapted to engage a surface of the sheet material, at least one resiliently deformable arm attached to the body and radially bendable relative to the body for facilitating introduction of the body into the opening and locking of the body in the opening, and a deformable film surrounding the arm. The film is tied on both the body and the flange portion to seal the opening and deformed concurrently with the deformation or bending of the arm. A locking portion is provided on the arm to engage an edge of the opening when the body is fit into the opening with a snapping action of the arm.

This invention relates to a clip-like member of plastic material.
It is specifically in the automotive industry that lid-like or stopper-like members are used and are inserted into openings in the sheet metal material. It is known to configure such members like clips. In other words, they have resilient snapping portions or the like which are adapted to engage the opposed edge of an opening in order to secure the member in its seat. For instance, it is known to mount tank troughs of plastic material in an appropriate receptacle opening of the automotive body. It is also known to sealingly close holes which serve for making car paint flow out, for instance, with a lid-like stopper subsequently. Such a hole stopper also is mostly formed as a clip.
A low mounting force is aimed at for such members which are lockable via snap-in connections. A high retaining force needs to be achieved at the same time. It is true that the members which are known may be dismounted in most cases. However, since the locking or snapping portion is mostly located on that side of the metal sheet which is inaccessible the member requires to be destroyed in order to dismount it. Then, it will be impossible to use it again.
In many cases, the members are intended to be sealedly seated in the associated opening. It is known to apply extra sealing material to between a flange portion and that area of the carrier which surrounds the opening. However, a separate sealing member will increase the expenditure. It would also be imaginable to adhere a sealing ring or the like to the flange portion. This increases the expenditure as well. For reasons of stability or other reasons, it is mostly desirable that the member be made of relatively solid or hard material. Hard material, however, is difficult to use for realizing sufficient sealing.
It is the object of the invention to provide a clip-like member which can be sealingly inserted into an opening and is easily mountable and dismountable.
The object is achieved by the features of claim 1.
In the inventive clip-like member, a body is provided which is designed to be fluid-tight in an axial direction. A body of this type can be a lid which closes an opening, e.g. one in an automotive body metallic sheet, or a stopper. For instance, holes which allow car paint to flow out and through which car paint will exit after the painting operation are provided in the automotive body. These have to be tightly closed subsequently for reasons of anti-corrosive protection. The body may also be formed by a tank trough which is snapped into the skin of the automotive body. The feature which all bodies have in common is that they are particularly fluid-tight, on their part.
Another feature of the inventive member is that it can be snapped in by means of at least one snapping portion which is formed to the circumference of the body and is elastically deformable relative to the body. The snapping portion has a locking portion which engages the edge of the opening after undergoing temporary deformation and/or warping of the snapping portion while the body is inserted into the opening of the side opposing the flange portion. However, it is also known as such how to mount a body in a hole or opening in such a manner. The fact essential to the invention is that a thin, easily deformable portion which interconnects the locking portion with the body is provided between the snapping portion and the body or the flange portion. The thin, deformable portion is of a continuous shape and, thus, forms a closed portion between the flange portion and the body in the area of the snapping portion.
The fact that the thin portion can be easily deformed and, thus, is also deformed when the snapping portion is temporarily deformed while the body is being inserted does not noticeably increase the insertion force of the member. Therefore, the snapping portion can be designed in such a way that it requires a small mounting force, on one hand, but produces a large retaining force, on the other. Nevertheless, if such a construction is used a complete sealing by the member is obtained in the opening.
Although it is imaginable to form the film or thin portion, which provides for the member to be sealed, from the same material as that of the body. However, according to an aspect of the invention, it is an advantage if the body and the snapping portion are molded from a first material which is relatively hard and the thin portion is formed from a second, elastomeric material in a two-component injection molding step. An elastomeric material has the advantage that it may readily be folded or stretched depending on the direction of stress while the snapping portion undergoes deformation.
Another aspect of the invention provides that a handle portion of the snapping portion projects beyond the thin portion on the same side as the flange portion. In this way, it is possible to dismount the member, starting from the flange side. Any accessibility from the opposite side is unnecessary for disassembly.
The invention is particularly advantageous in an embodiment in which the snapping portion is formed as an arm which is formed to the outer circumference of the body at an axial distance from the flange portion, the arm having a locking shoulder at the outer side thereof which engages the edge of the opening. Mounting it merely requires to pivot the arm, which action requires relatively small forces. When in the mounted position, the arm is acted on by pressure when the member is loaded in the extracting direction. The arm is capable of absorbing a significant stress by pressure before it becomes possible to remove the member from the opening. However, it can be easily dismounted by simply pivoting the arm over the handle portion.
According to a further aspect of the invention, in order to provide tightness between the flange portion and the carrier material as well, it might be an advantage if a sealing portion made of an elastic material, which sealingly engages the associated carrier surface, is molded to the flange portion in a two-component injection mold method.


Disclosed is an aspherical lens module of a vehicle headlamp configured such that a position light function is added to the existing functions of a low beam or high beam without increasing the size of the lamp module. Further, the aspherical lens module of a headlamp is configured such that the quantity of required light of a low beam or high beam is not reduced when the position light function is added to them.

Field of the Disclosure
The present disclosure relates to aspherical lens modules for vehicle headlamps.
Description of the Related Art
Generally, vehicles are provided with lamps to allow objects ahead of the vehicle to be seen during times of poor or reduced visibility, such as at night. Vehicle lamps also inform other vehicles or people using the road of the vehicle's presence. Headlamps, also referred to as headlights, light the road in front of vehicles and require enough brightness to identify obstacles on the road 100 m away during times of poor or reduced visibility, such as nighttime.
Conventional vehicle headlamps generally include a projection module mounted to a housing and a lens coupled to a circumferential surface of the housing. The projection module typically includes a reflector having a bulb and an aspherical lens provided in front of the reflector that emits light.
When power is supplied to a light source in the housing to turn the light source on, light generated from the light source is reflected by the reflector and emitted through an aspherical surface of the aspherical lens in a forward direction.
However, conventional aspherical lenses are not formed to be any particular shape and only function to emit light from a single light source through an elliptical optical system in a forward direction. Accordingly, sizes of lamp modules are typically increased in order to accommodate a position light function in addition to a low beam, a high beam, and a turn signal. Position lights may also be referred to as parking lights or daytime running lights. As a result, when a portion of the high beam reflector is used as the position light, high beam performance may be reduced.
The foregoing is intended merely to aid in the understanding of the background of the present disclosure. It is not intended to mean that the present disclosure falls within the purview of prior art that is already known to those of ordinary skill in the art.


A vibrating member made of a thin metal plate and adapted to be used in an acoustic transducer such as a loudspeaker or microphone is diffused at least partially with an inorganic substance to improve the acoustic characteristics thereof, particularly the high frequency range.

The vibrating member for use in an acoustic transducer, such as a diaphragm of a loudspeaker or a microphone, or a cantilever of a pickup cartridge of a disc-record player, is usually constructed of aluminum or titanium etc. which is light in weight and easily formable.
For the diaphragm of aluminum or titanium, there is a vibration mode produced which is spread throughout the diaphragm and provides a unique peak in its high frequency characteristics causing the reproduced sound quality to be considerably degraded.
When the vibrating member is a cantilever, it occupies a major part of the effective mass of the vibration system, so that it becomes difficult to minimize the effective mass of the vibration system to improve the performance of the cartridge except by reducing the mass of the cantilever. However, if the wall thickness and the diameter of the cantilever are reduced to minimize the weight thereof, the rigidity thereof is necessarily reduced causing the overall characteristics of the cantilever to be degraded.
In order to overcome the above mentioned disadvantages of the conventional vibrating member, an inorganic material having a large ratio of Young's modulus E to density .rho. should be used in fabricating the vibrating member. Examples of such material include boron, beryllium etc. Boron, particularly of high purity, is relatively difficult to obtain and beryllium can not be prepared without incurring large expenses to install equipment for preventing public pollution. Moreover, boron and beryllium are difficult to form mechanically. It has been proposed to form a vibrating member consisting solely of boron or beryllium, and thereby avoid the latter difficulty, by forming a coating layer of beryllium or boron on a suitably configured substrate of easily formable material and thereafter separating the coating layer from the substrate. The coating layer thus obtained is ready to use as a diaphragm or the like according to the specific shape of the substrate. However, the coating layer which would be provided by a vapor-deposition process is mechanically fragile. Moreover, an expensive installation, including electron beam apparatus and other equipment, is necessary to provide such coating on the substrate. In addition to this, the time required to provide the coating is considerably long. Therefore, the use of boron or beryllium, alone, for mechanically forming the vibrating member has not been practiced.
Therefore, it had been proposed to use aluminium or titanium, which is easily formable, as a substrate of the vibrating member and to coat the substrate with boron or beryllium by means of physical evaporation or chemical processing to form a coating layer. As is well known, in order to obtain good properties of the deposited layer and a good adhesion thereof to the substrate, it is desirable to heat the substrate to a temperature higher than 150.degree. C. However, since the thermal expansion coefficiencies of the coating layer and the substrate are usually very different, there is a strong possibility of mechanical deformation of the substrate and/or cracking of the coating layer by a subsequent cooling to the normal temperature after the heating and deposition process.

Vibrating member for acoustic transducer and method for manufacturing the same