PODOSOMES AS A THERAPEUTIC TARGET FOR INTRACRANIAL ANEURYSM

Abstract

Disclosed here are uses of anti-podosome therapies for treating a subject at risk of suffering from an aneurysm. The present disclosure demonstrates that endothelial podosomes plays a role in THSD1-mediated focal adhesion stability and aneurysm formation and characterizes molecular targets for therapeutic intervention.

Description

FIELD OF THE INVENTION

The present disclosure relates to the characterization of molecular targets for treating intracranial aneurysms (IA). Intracranial aneurysm (IA) is a cerebrovascular disease that predominantly occurs in the cerebral artery and is characterized by pathologic dilatation of blood vessels. Each intracranial aneurysm (IA) is a weakened area in a cerebral artery wall that leads to abnormal dilatation and rupture causing subarachnoid hemorrhage (SAH), a major cause of hemorrhagic stroke. A rupture of IA induces a subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke that frequently leads to death or severe disability. Due to early age of onset and high mortality, SAH accounts for >25% of years lost for all stroke victims under the age of 65 years. Despite treatment advances, SAH mortality rate is 40% and only half of survivors return to independent life.

There is a critical unmet need for understanding the genetic and molecular basis for IA to improve clinical outcomes through early therapeutic intervention.

SUMMARY

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

In some aspects, the present disclosure demonstrates that endothelial podosomes are regulated by the transmembrane receptor protein THSD1. THSD1 has been shown to have a pathological role in intracranial aneurysm disease. Briefly, the present disclosure provides that endothelial podosomes are novel therapeutic targets for the treatment, modulation, and prevention of aneurysm rupture. There is thus a need in the art for understanding the molecular mechanisms driving aneurysms. The present disclosure addresses this need.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A (FIG. 1A) illustrates simplified pedigrees of three IA families (NR4748, NR4931, and NR5704). Specifically, FIG. 1A illustrates Intracranial Aneurysm (IA) affected (black), aortic aneurysm (AA) affected (half black), unaffected individuals >45 (white), and individuals with unknown/unclear status (gray symbols).

FIG. 1B (FIG. 1B) depicts patient-identified THSD1 variants identified in the pedigrees of the families from FIG. 1B. The black bar represents the 853 amino acids of the WT version of the THSD1 gene. The diagram lists 8 variants found in patients affected by IA and AA, namely LSF, R450X, R460W, E466G, G600E, P639L, T6531, S775P. The back boxes illustrate the relative position of the signal peptide (SP), thrombospondin type 1 domain (TSP1), and transmembrane (TM) domains of THSD1.

FIG. 2 (FIG. 2) is a drawing depicting an intracranial aneurysm. An intracranial aneurysm, also known as a brain aneurysm, is a cerebrovascular disorder in which weakness in the wall of a cerebral artery or vein causes a localized dilation or ballooning of the blood vessel.

FIG. 3 (FIG. 3) is a drawing depicting a model of SRC-mediated podosome formation and ECM degradation. FIG. 3 schematic model of podosome illustrates an actin-enriched core surrounded by an adhesive ring.

FIG. 4a through FIG. 4b (FIGS. 4a-4b) are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation.

FIG. 5a through FIG. 5b (FIGS. 5a-5b) are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function.

FIG. 6a through FIG. 6b (FIGS. 6a-6b) are experimental results depicting that loss-of-function of THSD1 increase MT1-MMP cell surface exposure.

FIG. 7a through FIG. 7b (FIGS. 7a-7b) are experimental results depicting that loss-of-function of THSD1 activates MMP2.

FIG. 8a through FIG. 8b (FIGS. 8a-8b) are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish.

FIG. 9 (FIG. 9) are experimental results depicting that loss-of-function of Thsd1 promotes IA formation in circle of Willis in mice.

FIG. 10a through FIG. 10b (FIGS. 10a-10b) are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice.

FIG. 11 is a protein alignment between human and zebrafish THSD1.

FIG. 12 is a sequence alignment illustrating three rare variants of thsd1 conserved between humans and fish.

FIG. 13 is a graph charting the results of treatment of a model organism engineered with conserved mutations demonstrating that SRC inhibitors rescued intracranial hemorrhage. Three exemplary inhibitors disclosed on this chart include PP2, SU5565, and dasatinib.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTIONS

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

The rupture of an intracranial aneurysm frequently causes a subarachnoid hemorrhage (SAH), a type of stroke characterized by high morbidity and mortality. Specifically, the present disclosure demonstrates with data from three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status.

Previously, it has been reported that deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants caused disease in both familial and sporadic cases with supporting evidence from animal models. Of note, whole exome sequencing of large IA families identified (some members of the affected family are shown in the pedigree of FIG. 1A) a THSD1 nonsense mutation that segregated in all nine (9) affected and was absent in 13 unaffected family members. It was further discovered that eight (8) THSD1 rare missense variants in 507 unrelated patients/probands where each perturbed THSD1 cell adhesion activity.

These rare variants were highly enriched in case-control studies in comparison to ethnically matched controls. It was found that Thsd1 loss-of-function leads to brain hemorrhage and premature death in both zebrafish and mice. Further, Thsd1 heterozygous and null mice developed IA and suffered SAH. The study further demonstrated that THSD1 is highly expressed in endothelial cells of the cerebrovasculature, is important for cell adhesion, promotes nascent focal adhesion assembly via Talin interactions, and potentially regulates downstream signaling. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference in their entireties). However the study did not provide any insights on the THSD1 molecular pathways.

To further study the role of THSD1 in IA/SAH, additional analysis of whole exome sequencing of the IA families described in FIG. 1 was conducted. The detailed analysis identified a THSD1 nonsense mutation that segregated in all 9 affected and was absent in 13 unaffected family members. Notably, the exome sequencing analysis uncovered multiple signaling pathways that appeared to be affected by THSD1 expression: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling as well as TGFβ signaling (see Table 1, discussed in Example 1). The present disclosure contemplates that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression.

In some aspects, the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or function. In many instances the compound is a Src inhibitor, such as PP2. In some instances, the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib. In some instances the aneurysm is an intracranial aneurysm. In other instances the aneurysm is an aortic aneurysm. In many instances the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. The variant can be in a coding region of the THSD1 gene, in a control sequence of a non-coding region of the THSD1 gene, or in any other suitable region. In some instances, the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele, such as the exemplary variants identified and described in FIG. 1B, which include single codon substitutions at codon LSF, R460W, E466G, G600E, P639L, T6531, S775P.

In many instances, the compound that modulates podosome formation or function can be administered systemically. Systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal administration. In many instances the compound that modulates podosome formation or function is administered locally. Local administration can include, but is not limited to, (i) intracranial; (ii) intra-ocular; (iii) intra-nasal; (iv) intrathecal or (v) intra-vescular administration. In many instances the subject is a human.

In some aspects the disclosure provides an inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.

In some aspects the disclosure provides an inhibitor of podosome function for use in a method of treating an aneurysm in a subject.

In other aspects the disclosure provides a kit comprising an inhibitor of podosome formation or function described herein and instructions for use thereof.

Podosomes

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to, and degradation of, the extracellular matrix. A podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM (FIG. 3). Unlike other adhesive structures such as focal adhesion complexes that tether the endothelium to the ECM, the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9. Key proteins in these structures include the actin regulators cortactin and (N)-WASP, the adaptor proteins Tks4 and Tks5, and the metalloprotease MT1-MMP. Among all the upstream events that occur during podosome formation, the best characterized is the activation of SRC, a non-receptor tyrosine kinase. When SRC is enriched and activated in pre-podosome structures, it phosphorylates a wide array of substrates. For instance, SRC phosphorylates cortactin and promotes actin assembly in podosomes. Many cell types elaborate these structures, including vascular smooth muscle and endothelial cells. The present disclosure demonstrates that podosomes are also present in aneurysms.

Since intracellular matrix degradation is a hallmark of aneurysm development the present disclosure contemplates that modulation of podosome formation and function can provide a target for treating an aneurysm.

Podosomes are intimately involved in cell motility, with podosomes specifically implicated in cell invasion. Invasiveness is achieved through the secretion of matrix metalloproteinases (MMPs) from the core of podosomes, which degrade the extracellular matrix (ECM). This promotes motile behaviors that aid a range of processes including migration of aortic endothelial cells for arterial vessel remodeling. In some aspects, the

In addition, the initiation of podosome formation has been shown to be dependent on the underlying matrix, both in terms of its nature (which ligands are present) and its geometry (whether the ligands are uniformly distributed or arranged in subcellular-sized islands). Cells use different integrin receptors to detect the mechanical constraints of their environment and to decide accordingly whether a podosome should be initiated or not. Following initiation, substrate stiffness continues to play a role in the lifespan of the podosome, with increased stiffness resulting in increased longevity and decreased distance between individual podosomes. The present disclosure demonstrates that THSD1 knockdown in endothelial cells increases the expression of metalloprotease and evaluates the role of podosome formation and function in managing podosome formation in subjects, particularly in subjects afflicted with loss-of-function variants of THSD1 gene.

Podosome Inhibitors

The present disclosure describes the identification and characterization of genes whose perturbation leads to intracranial aneurysms (IA) and/or vascular endothelial cell dysfunction. Further, the present disclosure provides a characterization of molecular pathways that can provide novel therapeutic targets for treatment of aneurysms, particularly IA.

In some aspects the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or podosome function.

In some instances the compound is a Src inhibitor such as PP2.

In other instances the compound is SU6656. SU6656 was initially identified as a Src kinase inhibitor by virtue of its ability to reverse an effect that an activated mutant form of Src (hu SRC Y530F) has on the actin cytoskeleton, namely the formation of podosome rosettes, otherwise known as invadopodia. SU6656 was initially published as a Src family kinase inhibitor with selectivity relative to Platelet-derived growth factor receptor Tyrosine kinase. Subsequent studies have confirmed that SU6656 is relatively selective for Src family kinases.

In other instances the compound is KX2-391. KX2-391, also known as KX-01, is a first-in-class dual Src kinase and tubulin polymerization inhibitor that is currently only developed as a topical medicinal product.

In other instances the compound is saracatinib. Saracatinib is an experimental drug that acts as a dual kinase inhibitor, with selective actions as a Src inhibitor and a Bcr-Abl tyrosine-kinase inhibitor.

In other instances the compound is bosutinib. Bosutinib is a small molecule BCR-ABL and src tyrosine kinase inhibitor currently used for the treatment of chronic myelogenous leukemia.

In other instances the compound is dasatinib is a selective tyrosine kinase receptor inhibitor.

In other instances the compound is ponatinib. Ponatinib (also known as AP24534) is a potent, orally active tyrosine kinase inhibitor.

Methods of Treating Aneurysms

The methods, compositions, and uses of this disclosure may comprise a treatment method to arrest, reverse, or ameliorate an aneurysm, e.g., an intracranial aneurysm. In some cases, the therapeutic effect is achieved by administrating a therapeutically-effective dose of a compound that modulates podosome formation and function. Exemplary compounds contemplated by the disclosure include SU6656, a selective SRC inhibitor, KX2-391 and saracatinib (currently in clinical trials), and three FDA-approved compounds including, bosutinib, dasatinib, and ponatinib.

The treatment may comprise treating a subject (e.g. a patient at risk of having an intracranial aneurysm due to the presence of a THSD1 genetic variant or an animal with a similar genetic variant). The disease may be a weakness in a blood vessel in the brain that balloons and fills with blood, for example, a brain aneurysm (also called a cerebral aneurysm or an intracranial aneurysm) is a ballooning arising from a weakened area in the wall of a blood vessel in the brain. The subject may be a human.

Treatment may be provided to the subject before clinical onset of disease. For instance, in specific cases, treatment may be provided upon the identification of a THSD1 variant in a subject, before the onset of a disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. Because of the genetic aspect of IA, treatment may be provided through the lifetime of a subject that is afflicted with a THSD1 variant that may lead to subarachnoid hemorrhage. In some aspects, treatment will be prescribed to prevent IA in a subject that carries a THSD1 variant associated with IA.

A treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure. A treatment can comprise modulating the levels of podosomes in vivo. A treatment may comprise administering a suitable level of an autophagy inhibitor for reducing bulge's in the wall of a blood vessel and preventing Subarachnoid hemorrhage.

Further, there are many risk factors for the development of intracranial aneurysms, both inherited and acquired. Females are more prone to aneurysm rupture, with SAH times more common in women. The prevalence of aneurysms is increased in certain genetic diseases; the classic example is autosomal dominant polycystic kidney disease (ADPKD), but other diseases such as Ehlers-Danlos syndrome, neurofibromatosis, al-antitrypsin deficiency also demonstrate a link.

In ADPKD, 10% to 15% of patients develop intracranial aneurysms. Marfan's Syndrome was once thought to be linked to intracranial aneurysm formation, but recent evidence suggests that this may not be true. Aneurysms also run in families in the absence of an identified genetic disorder, with a prevalence of 7% to 20% in first or second degree relatives of patients who have suffered a SAH. FIG. 1A discloses a large family with variants (FIG. 1B) in the THSD1 gene that have been causally linked to IA.

Definitions

All of the functionalities described in connection with one embodiment of the methods, devices or instruments described herein are intended to be applicable to the additional embodiments of the methods, devices and instruments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of steps, components, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.

As used in the specification and claims of this application, the term “administering” includes any method which is effective to result in delivery of an autophagy inhibitor to the subject.

As used in this specification, the term “aneurysm” refers to broad classes of aneurysm, including aneurysms: abdominal aortic, thoracic aortic, and cerebral.

As used in this specification, the term “cerebral aneurysm” or “intracranial aneurysm” (also known as a brain aneurysm) is a weak or thin spot on an artery in the brain that balloons or bulges out and fills with blood. The bulging aneurysm can put pressure on the nerves or brain tissue. It may also burst or rupture, spilling blood into the surrounding tissue (called a hemorrhage). An unruptured aneurysm usually causes no symptoms. A key symptom of a ruptured aneurysm is a sudden, severe headache. Treatments for an unruptured aneurysm include medications to control blood pressure and procedures to prevent a future rupture.

As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.

As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.

As used in this specification, the term “thoracic aortic” aneurysm is an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel. A thoracic aortic aneurysm occurs in the part of the body's largest artery (the aorta) that passes through the chest.

As used in the specification and claims of this application, the term “at risk” or more specifically a “subject at risk of developing an intracranial aneurysm” is a subject afflicted with a genetic variant, e.g., THSD1 variant, that causes the subarachnoid hemorrhage seen when an aneurysm ruptures.

The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

EMBODIMENTS

EMBODIMENT 1. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation.

EMBODIMENT 2. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome function.

EMBODIMENT 3. The method of any one of embodiments 1 or 2, wherein the compound is a Src inhibitor.

EMBODIMENT 4. The method of embodiment 3, wherein the Src inhibitor is PP2.

EMBODIMENT 5. The method of any one of claim 1 or 2, wherein the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.

EMBODIMENT 6. The method of embodiment 5, wherein the compound is SU6656.

EMBODIMENT 7. The method of embodiment 5, wherein the compound is KX2-391.

EMBODIMENT 8. The method of embodiment 5, wherein the compound is saracatinib.

EMBODIMENT 9. The method of embodiment 5, wherein the compound is bosutinib.

EMBODIMENT 10. The method of embodiment 5, wherein the compound is dasatinib.

EMBODIMENT 11. The method of embodiment 5, wherein the compound is ponatinib.

EMBODIMENT 12. The method of any one of embodiments 1-5, wherein the aneurysm is an intracranial aneurysm.

EMBODIMENT 13. The method of any one of embodiments 1-5, wherein the aneurysm is an aortic aneurysm.

EMBODIMENT 14. The method of any one of embodiments 1-13, wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

EMBODIMENT 15. The method of embodiment 14, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

EMBODIMENT 16. The method of embodiment 14, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

EMBODIMENT 17. The method of embodiment 14, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.

EMBODIMENT 18. The method of embodiment 14, wherein the single codon substitution in the at least one THSD1 allele is at codon LSF, R460W, E466G, G600E, P639L, T653I, S775P.

EMBODIMENT 19. The method of any one of embodiments 1-18, wherein the compound that modulates podosome formation or function is administered systemically.

EMBODIMENT 20. The method of embodiment 19, wherein systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal.

EMBODIMENT 21. The method of embodiment 1, wherein the compound that modulates podosome formation or function is administered locally.

EMBODIMENT 22. The method of embodiment 21, wherein local administration includes, but is not limited to, (i) intracranial; (ii) intra-ocular (iii) intra-nasal; (iv) intrathecal or (v) intra-vascular.

EMBODIMENT 23. The method of embodiment 1, wherein the subject is a human.

EMBODIMENT 24. An inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.

EMBODIMENT 25. An inhibitor of podosome function for use in a method of treating an aneurysm in a subject.

EMBODIMENT 26. A kit comprising an inhibitor of podosome formation or function of any one of embodiments Error! Reference source not found.-Error! Reference source not found. and instructions for use thereof.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

The practice of some molecular techniques described herein may employ, unless otherwise indicated, techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such techniques and descriptions can be found in standard laboratory manuals such as Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.; all of which are herein incorporated in their entirety by reference for all purposes.

EXAMPLE 1: Molecular Dissection of THSD1 Function in Vascular Endothelial Cells from Global Transcriptomics

Genetic factors play a significant role in IA pathogenesis as illustrated by family studies and several IA predisposing syndromes. 7%-20% of all patients have a known family history and a family history is the strongest risk factor for disease. Excluding syndromes that account for less than 1% of all IA cases, candidate IA genes have been primarily identified by genome-wide association studies and more recently, by whole exome sequencing in affected families. Yet, little is known about the genetic causes of IA providing minimum insight for the understanding and development of therapeutic targets that could treat the disease.

Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants. FIG. 1A illustrates a large family pedigree studied as part of the present disclosure to identify novel candidate genes whose rare variants cause intracranial aneurysm. including NR4748, NR4931, NR5704.

The subject highlighted participated in the sequencing studies described herein. We studied three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status. Specifically, 21 individuals provided samples for sequencing for family NR4748. 32 individuals provided samples for sequencing for family NR4931. 16 individuals provided samples for sequencing for family NR5704.

Deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants cause disease in both familial and sporadic cases with supporting evidence from animal models. THSD1 is predominantly expressed in vascular endothelial cells. The work identified deleterious variants in thrombospondin-type 1 domain-containing protein 1 (THSD1) that can cause IA and SAH. Initial characterization of Thsd1 in two vertebrate models including zebrafish and mice lead to the discovery that THSD1 mediated cerebral hemorrhage is located in subarachnoid space in mice. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference).

However, the mechanism of action utilized by the discovered THSD1 variants to drive disease remained elusive. Further, there was little information describing genes and pathways regulated by THSD1 using global transcriptomics that could be used to inform the mechanism of action of THSD1. Thus, on its own the identification of THSD1 in the context of AI was not sufficient to inform a therapeutic strategy.

The present disclosure contemplated that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression. The present disclosure provides results from global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown that identifies THSD1-regulated specific genes and pathways that are critical for mediating its function, providing potential targets for therapeutic intervention in IA.

The instant disclosure provides RNAseq experiments in two THSD1 knock-down endothelial cell lines. The RNAseq results from both cell lines support the evidence that THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling. A few of these pathways were selected for further analysis and characterization.

Materials and Methods

Cell Culture

HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.

Knock-Down Experiments

Knockdown experiments in human vascular endothelial cells were performed using two distinct cell lines [HUVECs and Human brain microvascular endothelial cells(HBMECs)] using four siRNAs (two control siRNAs and two THSD1-specific siRNAs) to minimize erroneous findings due to off-target effects.

Transcriptome Profiling

Bioinformatic analyses of the global transcriptome were performed on rRNA-depleted RNA samples by RNA-Seq. Table 1 illustrates results of the analysis. As shown on Table 1, THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling) as well as TGFβ signaling.

TABLE 1
Statistically Significant THSD1-Regulated Pathways
(p-value < 0.005; Efron-Tibshirani's GSA test)
Description
Integrin Signaling
PI3K/Akt/mTOR Signaling
Src Signaling
Rho Signaling
Tgfβ

We identified a number of genes that are affected by the lack of THSD1 in the knock-down cell lines and are likely regulated by THSD1. A subset of these genes likely contributes to disease pathobiology and may be targets for therapeutic intervention. Table 2. Describes genes differentially expressed in THSD1 knockdowns.

TABLE 2
Genes differentially expressed in THSD1 knockdowns; human
brain microvascular endothelial cells and HUVECs, combined
Description             Genes
Differential Expression ALOX5AP, ARL17B, CTSS, GUK1,
upon THSD1 Loss         IFNAR2, LAMC2, NHLRC4, NISCH,
Upregulated genes       OLFML3, PSMD11, PUM2, SLC37A1,
                        SMAP1, SNORA27, TMEM50B, UBAP2,
                        ZNF780A
Differential Expression ABCG2, ADAMTS1, AQP1, BCAP31,
upon THSD1 Loss         CDC45, CHCHD7, DMD, ENOX2,
Downregulated genes     FAM219A, FGF2, FGFR1OP2, G0S2, GJA4,
                        IGFBP3, INAFM1, MAP2, MGP, NOL3,
                        NPR1, NVL, PPHLN1, SERPINB2, SETBP1,
                        (THSD1), TMEM107, ZNF185

Table 3. lists genes differentially expressed in THSD1 knockdown HUVECs.

TABLE 3
Differentially Expressed Genes in THSD1 Knockdown HUVECs
Ensembl Gene	Gene Name	logFC	logCPM	LR	PValue	FDR
ENSG00000136114	THSD1	−2.07	4.85	141.12	1.52E−32	2.15E−28
ENSG00000265107	GJA5	−3.81	3.42	137.13	1.13E−31	8.01E−28
ENSG00000130066	SAT1	−1.74	8.37	128.15	1.04E−29	4.92E−26
ENSG00000111341	MGP	−1.46	6.50	112.09	3.42E−26	1.21E−22
ENSG00000187513	GJA4	−2.29	4.32	102.47	4.39E−24	1.25E−20
ENSG00000284057	AP001273.2	8.67	−0.99	71.34	3.00E−17	7.11E−14
ENSG00000170323	FABP4	−2.09	6.36	66.29	3.90E−16	7.91E−13
ENSG00000125266	EFNB2	−1.28	7.77	63.28	1.79E−15	3.18E−12
ENSG00000113389	NPR3	−3.16	1.25	56.87	4.65E−14	7.35E−11
ENSG00000134668	SPOCD1	1.16	5.45	54.03	1.97E−13	2.80E−10
ENSG00000118777	ABCG2	−2.38	4.92	51.45	7.36E−13	9.50E−10
ENSG00000203812	HIST2H2AA3	4.21	0.20	50.59	1.14E−12	1.35E−09
ENSG00000272921	AC005832.4	−8.85	−0.72	48.91	2.69E−12	2.93E−09
ENSG00000078018	MAP2	−1.06	4.84	48.51	3.29E−12	3.34E−09
ENSG00000115884	SDC1	−1.53	3.23	48.33	3.61E−12	3.42E−09
ENSG00000139278	GLIPR1	1.13	6.53	47.73	4.89E−12	4.34E−09
ENSG00000101335	MYL9	1.09	4.76	47.07	6.83E−12	5.71E−09
ENSG00000181634	TNFSF15	1.64	5.10	45.62	1.43E−11	1.13E−08
ENSG00000122861	PLAU	1.03	5.58	43.85	3.55E−11	2.65E−08
ENSG00000107551	RASSF4	−1.10	5.07	43.68	3.86E−11	2.74E−08
ENSG00000274611	TBC1D3	−7.97	−1.52	43.13	5.12E−11	3.46E−08
ENSG00000149591	TAGLN	1.81	2.54	42.71	6.35E−11	4.10E−08
ENSG00000171435	KSR2	1.18	3.90	38.39	5.80E−10	3.58E−07
ENSG00000153165	RGPD3	4.26	−1.48	37.47	9.26E−10	5.48E−07
ENSG00000251569	AC093899.2	7.32	−2.20	34.85	3.55E−09	2.02E−06
ENSG00000163083	INHBB	−2.57	2.18	34.75	3.75E−09	2.05E−06
ENSG00000272949	AC093668.2	−7.24	−2.15	33.67	6.51E−09	3.43E−06
ENSG00000152217	SETBP1	−1.78	3.75	33.17	8.42E−09	4.27E−06
ENSG00000119900	OGFRL1	0.85	6.59	32.79	1.03E−08	5.02E−06
ENSG00000082684	SEMA5B	−4.57	−1.62	32.23	1.37E−08	6.26E−06
ENSG00000169604	ANTXR1	0.98	5.91	32.24	1.36E−08	6.26E−06
ENSG00000137573	SULF1	−1.27	5.03	31.88	1.64E−08	7.29E−06
ENSG00000115232	ITGA4	1.35	3.29	30.82	2.83E−08	1.22E−05
ENSG00000058085	LAMC2	1.06	5.71	30.05	4.21E−08	1.76E−05
ENSG00000022567	SLC45A4	−1.35	3.07	29.70	5.04E−08	1.99E−05
ENSG00000172602	RND1	−1.74	1.64	29.71	5.03E−08	1.99E−05
ENSG00000103426	CORO7-PAM16	4.38	−1.48	29.02	7.16E−08	2.75E−05
ENSG00000120217	CD274	1.35	4.21	28.77	8.14E−08	3.04E−05
ENSG00000116667	C1orf21	−0.99	4.42	27.88	1.29E−07	4.70E−05
ENSG00000169129	AFAP1L2	−2.16	3.16	27.67	1.44E−07	5.10E−05
ENSG00000138646	HERC5	−1.85	1.47	27.51	1.56E−07	5.29E−05
ENSG00000197646	PDCD1LG2	1.07	3.58	27.55	1.53E−07	5.29E−05
ENSG00000064651	SLC12A2	0.73	7.05	26.91	2.13E−07	7.03E−05
ENSG00000171388	APLN	0.85	9.85	26.02	3.39E−07	0.000109
ENSG00000175426	PCSK1	1.51	2.68	25.80	3.79E−07	0.00012
ENSG00000057019	DCBLD2	0.91	6.78	25.67	4.05E−07	0.000125
ENSG00000008517	IL32	0.69	6.11	25.52	4.37E−07	0.000132
ENSG00000138685	FGF2	−1.20	5.72	25.21	5.15E−07	0.000152
ENSG00000065308	TRAM2	0.67	8.10	25.04	5.60E−07	0.000162
ENSG00000137507	LRRC32	−0.87	7.22	24.74	6.57E−07	0.000187
ENSG00000129116	PALLD	0.69	6.27	24.56	7.22E−07	0.000201
ENSG00000129521	EGLN3	−1.85	0.09	24.44	7.66E−07	0.000209
ENSG00000125954	CHURC1-FNTB	−6.59	−2.67	24.25	8.46E−07	0.000227
ENSG00000152402	GUCY1A2	1.90	1.31	24.20	8.68E−07	0.000228
ENSG00000169429	CXCL8	1.22	4.62	23.66	1.15E−06	0.000297
ENSG00000164683	HEY1	−1.25	3.49	23.30	1.38E−06	0.000345
ENSG00000176907	TCIM	0.95	3.77	23.31	1.38E−06	0.000345
ENSG00000082126	MPP4	0.76	4.76	23.08	1.56E−06	0.000379
ENSG00000163637	PRICKLE2	−0.99	3.76	23.06	1.57E−06	0.000379
ENSG00000148143	ZNF462	0.72	4.95	22.92	1.69E−06	0.0004
ENSG00000139289	PHLDA1	0.63	7.00	22.22	2.43E−06	0.000566
ENSG00000028137	TNFRSF1B	−0.99	6.14	22.02	2.69E−06	0.000617
ENSG00000139629	GALNT6	0.66	5.98	21.94	2.82E−06	0.000635
ENSG00000117586	TNFSF4	0.66	7.43	21.80	3.03E−06	0.000672
ENSG00000256514	AP003419.1	1.68	0.91	21.47	3.60E−06	0.000787
ENSG00000240583	AQP1	−1.84	3.43	21.27	3.99E−06	0.000858
ENSG00000115252	PDE1A	−2.04	0.57	21.17	4.20E−06	0.00089
ENSG00000178695	KCTD12	−0.71	8.94	20.99	4.62E−06	0.000965
ENSG00000173535	TNFRSF10C	−0.65	6.69	20.89	4.87E−06	0.001002
ENSG00000156298	TSPAN7	−1.50	3.22	20.81	5.08E−06	0.001031
ENSG00000037280	FLT4	−0.95	6.22	20.66	5.48E−06	0.001066
ENSG00000118515	SGK1	1.02	6.19	20.70	5.37E−06	0.001066
ENSG00000171105	INSR	−0.82	5.20	20.67	5.47E−06	0.001066
ENSG00000128917	DLL4	−1.06	6.99	20.55	5.81E−06	0.001116
ENSG00000164284	GRPEL2	0.71	5.83	20.28	6.70E−06	0.001268
ENSG00000130449	ZSWIM6	0.84	6.22	20.20	6.96E−06	0.0013
ENSG00000165029	ABCA1	−1.20	4.86	20.18	7.06E−06	0.001303
ENSG00000143127	ITGA10	−0.95	4.84	20.08	7.43E−06	0.001336
ENSG00000187720	THSD4	0.77	5.68	20.08	7.43E−06	0.001336
ENSG00000073008	PVR	0.69	7.34	19.90	8.14E−06	0.001422
ENSG00000107731	UNC5B	−1.58	3.90	19.93	8.03E−06	0.001422
ENSG00000122786	CALD1	0.71	9.31	19.89	8.21E−06	0.001422
ENSG00000110092	CCND1	0.77	8.26	19.76	8.76E−06	0.0015
ENSG00000154734	ADAMTS1	−1.32	3.65	19.66	9.23E−06	0.001561
ENSG00000142178	SIK1	1.16	2.11	19.62	9.46E−06	0.001581
ENSG00000168843	FSTL5	1.55	0.35	19.53	9.92E−06	0.001638
ENSG00000121858	TNFSF10	−0.94	4.92	19.41	1.05E−05	0.001722
ENSG00000169213	RAB3B	0.71	5.47	19.35	1.09E−05	0.001754
ENSG00000166670	MMP10	1.61	6.00	19.29	1.12E−05	0.00179
ENSG00000138347	MYPN	2.35	−1.12	19.03	1.29E−05	0.002029
ENSG00000145247	OCIAD2	0.76	5.62	18.90	1.38E−05	0.002126
ENSG00000160801	PTH1R	−2.70	−1.67	18.89	1.38E−05	0.002126
ENSG00000184113	CLDN5	−0.91	7.73	18.88	1.39E−05	0.002126
ENSG00000152518	ZFP36L2	−0.62	6.87	18.76	1.48E−05	0.002235
ENSG00000171617	ENC1	0.87	6.92	18.56	1.64E−05	0.002457
ENSG00000166833	NAV2	0.61	5.37	18.42	1.77E−05	0.00262
ENSG00000177076	ACER2	−1.05	3.11	18.28	1.91E−05	0.002793
ENSG00000154928	EPHB1	1.07	2.28	18.16	2.03E−05	0.002915
ENSG00000162407	PLPP3	−0.99	5.19	18.16	2.03E−05	0.002915
ENSG00000099204	ABLIM1	−0.73	8.31	18.11	2.09E−05	0.002957
ENSG00000177606	JUN	0.68	7.59	18.09	2.10E−05	0.002957
ENSG00000170006	TMEM154	0.74	5.48	18.03	2.17E−05	0.003026
ENSG00000118762	PKD2	−0.61	5.46	17.91	2.32E−05	0.003197
ENSG00000108387	‘SEPT4	−1.64	−0.22	17.45	2.96E−05	0.004038
ENSG00000170891	CYTL1	0.91	3.83	17.25	3.28E−05	0.004439
ENSG00000175040	CHST2	0.88	4.58	17.17	3.42E−05	0.004583
ENSG00000167037	SGSM1	−1.41	1.33	17.15	3.45E−05	0.004587
ENSG00000185070	FLRT2	0.70	7.60	17.09	3.57E−05	0.004694
ENSG00000180304	OAZ2	−0.56	6.82	16.78	4.20E−05	0.005473
ENSG00000257093	KIAA1147	−0.59	7.24	16.71	4.36E−05	0.005628
ENSG00000184897	H1FX	−0.58	6.28	16.64	4.53E−05	0.005794
ENSG00000075426	FOSL2	0.57	6.66	16.52	4.80E−05	0.006094
ENSG00000105825	TFPI2	0.57	7.14	16.40	5.13E−05	0.006449
ENSG00000135318	NT5E	0.61	7.62	16.38	5.18E−05	0.00645
ENSG00000127533	F2RL3	1.23	1.06	16.34	5.30E−05	0.006492
ENSG00000205683	DPF3	1.21	2.98	16.34	5.30E−05	0.006492
ENSG00000105499	PLA2G4C	0.76	3.68	16.29	5.44E−05	0.006599
ENSG00000167984	NLRC3	0.99	2.82	16.02	6.26E−05	0.007507
ENSG00000168685	IL7R	1.30	1.79	16.00	6.32E−05	0.007507
ENSG00000172985	SH3RF3	0.64	5.05	16.00	6.34E−05	0.007507
ENSG00000108551	RASD1	1.21	2.55	15.95	6.49E−05	0.00762
ENSG00000081087	OSTM1	0.67	5.66	15.81	6.99E−05	0.008139
ENSG00000128849	CGNL1	−0.78	6.46	15.61	7.78E−05	0.008985
ENSG00000133816	MICAL2	0.69	7.58	15.59	7.87E−05	0.009013
ENSG00000132965	ALOX5AP	2.25	−1.61	15.55	8.04E−05	0.009132
ENSG00000084734	GCKR	−2.84	−1.71	15.36	8.89E−05	0.009794
ENSG00000108854	SMURF2	0.53	7.94	15.38	8.81E−05	0.009794
ENSG00000111859	NEDD9	0.52	6.27	15.36	8.89E−05	0.009794
ENSG00000154678	PDE1C	0.89	3.99	15.37	8.84E−05	0.009794
ENSG00000033867	SLC4A7	0.58	7.03	15.33	9.05E−05	0.009889
ENSG00000152207	CYSLTR2	−2.45	−0.56	15.25	9.42E−05	0.010156
ENSG00000188452	CERKL	1.26	0.91	15.25	9.44E−05	0.010156
ENSG00000105357	MYH14	1.43	0.19	15.20	9.66E−05	0.010323
ENSG00000185432	METTL7A	−1.01	3.66	15.17	9.83E−05	0.010418
ENSG00000004799	PDK4	−1.18	2.61	15.12	0.000101	0.010639
ENSG00000122694	GLIPR2	0.65	4.88	15.10	0.000102	0.010655
ENSG00000213402	PTPRCAP	−3.20	−2.40	15.08	0.000103	0.010665
ENSG00000115902	SLC1A4	−0.69	4.85	14.96	0.00011	0.011119
ENSG00000169248	CXCL11	1.35	0.90	14.97	0.000109	0.011119
ENSG00000189367	KIAA0408	1.83	−0.79	14.96	0.00011	0.011119
ENSG00000102755	FLT1	−0.77	8.53	14.90	0.000114	0.011449
ENSG00000135905	DOCK10	0.62	5.30	14.86	0.000116	0.01158
ENSG00000079102	RUNX1T1	−0.82	4.50	14.83	0.000118	0.011588
ENSG00000153208	MERTK	0.58	5.53	14.83	0.000118	0.011588
ENSG00000198435	NRARP	−0.63	5.05	14.82	0.000118	0.011588
ENSG00000141682	PMAIP1	1.02	4.25	14.80	0.00012	0.01165
ENSG00000011347	SYT7	−1.86	−0.09	14.71	0.000126	0.012144
ENSG00000135842	FAM129A	−1.24	1.66	14.52	0.000139	0.013339
ENSG00000020577	SAMD4A	0.62	5.94	14.47	0.000143	0.013504
ENSG00000143153	ATP1B1	0.74	5.54	14.47	0.000142	0.013504
ENSG00000177666	PNPLA2	−0.55	6.36	14.43	0.000145	0.013671
ENSG00000085276	MECOM	−0.53	7.33	14.39	0.000148	0.013802
ENSG00000111145	ELK3	−0.64	8.64	14.39	0.000149	0.013802
ENSG00000140675	SLC5A2	−5.88	−3.18	14.34	0.000152	0.014056
ENSG00000005108	THSD7A	−0.61	5.99	14.27	0.000159	0.014543
ENSG00000157613	CREB3L1	1.04	1.76	14.23	0.000162	0.014743
ENSG00000163584	RPL22L1	−0.61	5.30	14.18	0.000166	0.015011
ENSG00000169418	NPR1	−1.45	3.01	14.12	0.000171	0.015403
ENSG00000131016	AKAP12	0.59	10.29	14.07	0.000176	0.015751
ENSG00000141298	SSH2	0.55	6.75	13.98	0.000184	0.016299
ENSG00000163131	CTSS	0.96	2.75	13.98	0.000185	0.016299
ENSG00000026508	CD44	0.69	5.54	13.94	0.000189	0.016335
ENSG00000136404	TM6SF1	0.69	4.25	13.92	0.00019	0.016335
ENSG00000144583	‘MARCH4	0.82	5.53	13.95	0.000188	0.016335
ENSG00000175899	A2M	−1.56	2.41	13.92	0.000191	0.016335
ENSG00000176749	CDK5R1	1.01	2.89	13.94	0.000189	0.016335
ENSG00000258984	UBE2F-SCLY	3.28	−1.94	13.88	0.000194	0.016538
ENSG00000136960	ENPP2	−1.14	1.35	13.86	0.000197	0.016639
ENSG00000140416	TPM1	0.61	8.65	13.81	0.000202	0.016977
ENSG00000112541	PDE10A	−0.92	3.09	13.76	0.000208	0.017288
ENSG00000198286	CARD11	0.59	5.90	13.76	0.000208	0.017288
ENSG00000108825	PTGES3L-AARSD1	1.90	−0.91	13.67	0.000218	0.017886
ENSG00000185650	ZFP36L1	0.51	6.82	13.66	0.000219	0.017886
ENSG00000188290	HES4	−1.30	2.20	13.66	0.000219	0.017886
ENSG00000114631	PODXL2	0.66	4.05	13.62	0.000224	0.018189
ENSG00000106852	LHX6	0.94	4.20	13.59	0.000227	0.018332
ENSG00000171115	GIMAP8	−0.63	7.66	13.56	0.000231	0.018514
ENSG00000189120	SP6	1.04	1.77	13.54	0.000234	0.018568
ENSG00000265972	TXNIP	−1.35	3.74	13.54	0.000234	0.018568
ENSG00000154639	CXADR	0.57	4.98	13.51	0.000237	0.018606
ENSG00000169242	EFNA1	−0.75	6.23	13.52	0.000237	0.018606
ENSG00000204304	PBX2	−0.72	5.80	13.49	0.00024	0.018755
ENSG00000157557	ETS2	0.56	6.59	13.45	0.000244	0.018975
ENSG00000105855	ITGB8	1.01	3.67	13.44	0.000246	0.018999
ENSG00000144802	NFKBIZ	0.88	4.33	13.37	0.000255	0.019615
ENSG00000204262	COL5A2	0.51	8.22	13.33	0.000262	0.019978
ENSG00000163092	XIRP2	2.56	−0.54	13.31	0.000264	0.020085
ENSG00000049540	ELN	−2.37	−1.72	13.23	0.000275	0.020704
ENSG00000166341	DCHS1	0.50	7.06	13.24	0.000274	0.020704
ENSG00000115414	FN1	0.69	12.30	13.17	0.000284	0.021259
ENSG00000094880	CDC23	−0.63	5.49	13.10	0.000295	0.021903
ENSG00000164647	STEAP1	−0.67	4.33	13.10	0.000296	0.021903
ENSG00000119681	LTBP2	0.48	9.54	13.03	0.000306	0.022511
ENSG00000107562	CXCL12	2.40	−1.46	12.98	0.000314	0.022927
ENSG00000138448	ITGAV	0.55	8.76	12.96	0.000318	0.022927
ENSG00000160223	ICOSLG	−0.72	3.61	12.95	0.00032	0.022927
ENSG00000163644	PPM1K	−0.64	3.93	12.96	0.000318	0.022927
ENSG00000186575	NF2	0.58	6.53	12.95	0.000319	0.022927
ENSG00000109906	ZBTB16	−1.08	1.12	12.94	0.000322	0.022986
ENSG00000144476	ACKR3	−0.77	4.06	12.89	0.00033	0.023468
ENSG00000136011	STAB2	−3.99	−2.80	12.86	0.000335	0.023698
ENSG00000074181	NOTCH3	−1.01	1.47	12.84	0.000338	0.023798
ENSG00000168916	ZNF608	−0.63	5.15	12.72	0.000362	0.025298
ENSG00000076351	SLC46A1	0.82	2.77	12.71	0.000364	0.025315
ENSG00000151474	FRMD4A	0.53	7.31	12.66	0.000374	0.025951
ENSG00000139508	SLC46A3	−1.45	4.08	12.58	0.00039	0.026876
ENSG00000131386	GALNT15	−1.70	0.16	12.53	0.000401	0.027499
ENSG00000101017	CD40	−0.65	4.42	12.49	0.000409	0.027854
ENSG00000270316	BORCS7-ASMT	−6.14	−3.00	12.49	0.00041	0.027854
ENSG00000141668	CBLN2	2.75	0.19	12.48	0.000412	0.027879
ENSG00000146072	TNFRSF21	0.47	6.64	12.44	0.000419	0.028234
ENSG00000183287	CCBE1	1.06	1.39	12.39	0.000432	0.028931
ENSG00000029534	ANK1	−1.37	0.01	12.37	0.000436	0.029068
ENSG00000124593	AL365205.1	−0.90	3.18	12.35	0.00044	0.029134
ENSG00000281028	AC104662.2	−6.09	−3.04	12.35	0.000441	0.029134
ENSG00000187942	LDLRAD2	−0.76	3.41	12.33	0.000446	0.029332
ENSG00000071246	VASH1	−0.66	7.76	12.30	0.000453	0.029655
ENSG00000269307	AC010463.1	−6.06	−3.06	12.27	0.000461	0.030027
ENSG00000050405	LIMA1	0.47	7.26	12.26	0.000463	0.030037
ENSG00000259171	AL163636.2	−1.37	1.58	12.25	0.000465	0.030049
ENSG00000048740	CELF2	0.49	6.23	12.18	0.000483	0.030936
ENSG00000116774	OLFML3	0.99	1.45	12.18	0.000482	0.030936
ENSG00000182240	BACE2	−0.56	7.52	12.17	0.000486	0.030971
ENSG00000204767	FAM196B	0.77	3.39	12.15	0.00049	0.031102
ENSG00000211448	DIO2	2.26	−1.84	12.14	0.000494	0.031198
ENSG00000151468	CCDC3	−1.17	0.72	12.11	0.000501	0.031481
ENSG00000196498	NCOR2	0.52	7.49	12.09	0.000506	0.031651
ENSG00000049130	KITLG	0.70	5.16	12.07	0.000512	0.031901
ENSG00000137033	IL33	−1.94	4.40	12.06	0.000516	0.031997
ENSG00000130635	COL5A1	0.52	8.58	12.03	0.000524	0.032215
ENSG00000133056	PIK3C2B	−0.67	5.90	12.03	0.000522	0.032215
ENSG00000116741	RGS2	0.79	3.02	12.02	0.000528	0.0323
ENSG00000129757	CDKN1C	−0.95	2.05	11.98	0.000536	0.032564
ENSG00000179195	ZNF664	−0.66	6.99	11.99	0.000535	0.032564
ENSG00000185924	RTN4RL1	−2.85	−2.54	11.97	0.000542	0.032704
ENSG00000189060	H1F0	−0.50	6.44	11.96	0.000543	0.032704
ENSG00000255767	AC108488.2	5.64	−3.35	11.95	0.000548	0.032834
ENSG00000108691	CCL2	0.56	5.05	11.89	0.000566	0.033765
ENSG00000106069	CHN2	0.83	2.52	11.87	0.000571	0.033914
ENSG00000132702	HAPLN2	1.07	3.28	11.85	0.000575	0.033914
ENSG00000183691	NOG	1.96	0.87	11.85	0.000575	0.033914
ENSG00000105738	SIPA1L3	0.53	5.37	11.84	0.000578	0.033924
ENSG00000154096	THY1	−1.73	0.37	11.84	0.00058	0.033924
ENSG00000140937	CDH11	0.58	5.50	11.80	0.000592	0.034495
ENSG00000127241	MASP1	−1.60	−0.50	11.77	0.000601	0.034815
ENSG00000164574	GALNT10	0.47	7.18	11.77	0.000603	0.034815
ENSG00000259112	NDUFC2-KCTD14	5.59	−3.38	11.75	0.000609	0.035044
ENSG00000109046	WSB1	−0.57	8.59	11.72	0.000619	0.035055
ENSG00000130054	FAM155B	1.73	−1.28	11.72	0.000619	0.035055
ENSG00000133401	PDZD2	−0.68	3.72	11.72	0.000618	0.035055
ENSG00000284041	AC073111.3	−5.87	−3.18	11.72	0.000619	0.035055
ENSG00000115008	IL1A	1.56	1.13	11.67	0.000634	0.035722
ENSG00000185737	NRG3	−0.86	2.50	11.66	0.000638	0.035834
ENSG00000196923	PDLIM7	0.57	7.15	11.64	0.000644	0.036026
ENSG00000187583	PLEKHN1	1.60	−1.07	11.62	0.000652	0.036337
ENSG00000135324	MRAP2	−0.97	3.21	11.57	0.000672	0.037286
ENSG00000158186	MRAS	0.56	4.27	11.55	0.000678	0.037503
ENSG00000092969	TGFB2	1.40	3.41	11.54	0.000682	0.037523
ENSG00000158373	HIST1H2BD	1.95	0.56	11.53	0.000684	0.037523
ENSG00000118946	PCDH17	1.00	3.06	11.49	0.0007	0.038238
ENSG00000067798	NAV3	0.64	5.24	11.48	0.000705	0.038357
ENSG00000143344	RGL1	0.81	6.48	11.46	0.00071	0.03848
ENSG00000188042	ARL4C	0.65	3.97	11.43	0.000723	0.039053
ENSG00000162772	ATF3	0.98	1.96	11.41	0.000731	0.039331
ENSG00000164104	HMGB2	−0.50	7.51	11.38	0.000742	0.039771
ENSG00000138411	HECW2	0.47	7.31	11.36	0.000749	0.03998
ENSG00000142627	EPHA2	0.49	7.57	11.33	0.000763	0.040464
ENSG00000258947	TUBB3	0.62	7.55	11.33	0.000761	0.040464
ENSG00000183775	KCTD16	1.30	−0.12	11.30	0.000773	0.040837
ENSG00000114948	ADAM23	0.55	5.15	11.26	0.000793	0.041703
ENSG00000114315	HES1	−0.49	5.56	11.22	0.000811	0.042315
ENSG00000138772	ANXA3	0.65	5.85	11.22	0.000808	0.042315
ENSG00000270276	HIST2H4B	−1.49	−0.13	11.21	0.000813	0.042315
ENSG00000213694	S1PR3	0.71	4.78	11.16	0.000835	0.043284
ENSG00000150687	PRSS23	0.44	9.70	11.15	0.000841	0.043462
ENSG00000095303	PTGS1	0.62	4.28	11.12	0.000852	0.043854
ENSG00000146674	IGFBP3	−0.93	1.59	11.11	0.00086	0.04411
ENSG00000109436	TBC1D9	0.45	6.66	11.09	0.000868	0.044376
ENSG00000274933	TBC1D3I	3.01	−2.32	11.07	0.000879	0.044738
ENSG00000116678	LEPR	−0.62	4.60	11.04	0.000893	0.044947
ENSG00000156642	NPTN	−0.65	6.37	11.04	0.000892	0.044947
ENSG00000198720	ANKRD13B	0.53	4.45	11.03	0.000895	0.044947
ENSG00000228144	AC078927.1	5.57	−3.50	11.04	0.000891	0.044947
ENSG00000256966	AL513165.2	2.50	−2.24	11.01	0.000907	0.045379
ENSG00000156920	ADGRG4	3.17	−2.89	10.97	0.000926	0.046138
ENSG00000171877	FRMD5	0.66	4.15	10.92	0.00095	0.047183
ENSG00000164946	FREM1	1.21	0.17	10.87	0.000975	0.048254
ENSG00000159640	ACE	−0.99	5.64	10.86	0.000982	0.048417
ENSG00000176771	NCKAP5	1.30	0.36	10.84	0.000993	0.048822
ENSG00000198513	ATL1	1.00	3.22	10.83	0.000999	0.048924
ENSG00000100234	TIMP3	−0.98	3.13	10.82	0.001006	0.049108

Table 4 lists genes differentially expressed in THSD1 knockdown HUVECs

TABLE 4
Differentially Expressed Genes in THSD1 Knockdown HBMECs
Ensembl Gene	Gene Symbol	logFC	logCPM	LR	PValue	FDR
ENSG00000117152	RGS4	−1.59	6.07	88.46	5.18E−21	7.37E−17
ENSG00000272949	AC093668.2	8.74	−0.80	64.63	9.02E−16	6.40E−12
ENSG00000283088	AC010487.3	−8.71	−0.85	60.76	6.45E−15	3.05E−11
ENSG00000136114	THSD1	−1.51	4.73	58.41	2.13E−14	7.56E−11
ENSG00000146674	IGFBP3	−2.59	2.50	54.84	1.31E−13	3.72E−10
ENSG00000284057	AP001273.2	8.19	−1.30	52.15	5.15E−13	1.22E−09
ENSG00000133101	CCNA1	−1.31	4.76	45.04	1.93E−11	3.92E−08
ENSG00000078018	MAP2	−1.31	4.98	43.56	4.11E−11	7.29E−08
ENSG00000240583	AQP1	−2.37	3.93	41.78	1.02E−10	1.61E−07
ENSG00000008517	IL32	1.06	6.45	39.73	2.92E−10	4.15E−07
ENSG00000154734	ADAMTS1	−1.55	3.82	38.76	4.78E−10	6.18E−07
ENSG00000068489	PRR11	−1.00	6.19	37.14	1.10E−09	1.30E−06
ENSG00000118777	ABCG2	−1.75	3.44	36.14	1.84E−09	1.87E−06
ENSG00000168542	COL3A1	2.68	1.64	36.27	1.72E−09	1.87E−06
ENSG00000138180	CEP55	−0.99	6.19	35.71	2.30E−09	2.17E−06
ENSG00000272414	FAM47E-STBD1	4.42	−1.05	35.52	2.52E−09	2.24E−06
ENSG00000264187	AC055811.2	7.52	−1.89	35.39	2.70E−09	2.26E−06
ENSG00000072571	HMMR	−1.09	5.70	34.95	3.39E−09	2.67E−06
ENSG00000112984	KIF20A	−1.03	6.21	34.59	4.07E−09	3.05E−06
ENSG00000126787	DLGAP5	−0.96	6.86	34.15	5.09E−09	3.62E−06
ENSG00000081087	OSTM1	1.10	5.56	33.06	8.93E−09	6.04E−06
ENSG00000117399	CDC20	−1.02	6.40	32.40	1.25E−08	8.08E−06
ENSG00000100292	HMOX1	0.94	6.83	31.53	1.96E−08	1.21E−05
ENSG00000134057	CCNB1	−0.94	6.95	31.40	2.10E−08	1.24E−05
ENSG00000143228	NUF2	−1.06	5.01	30.90	2.71E−08	1.48E−05
ENSG00000166851	PLK1	−1.03	6.17	30.91	2.70E−08	1.48E−05
ENSG00000108691	CCL2	1.38	5.33	29.45	5.74E−08	2.86E−05
ENSG00000132470	ITGB4	−1.94	3.69	29.41	5.84E−08	2.86E−05
ENSG00000164104	HMGB2	−0.86	7.35	29.54	5.47E−08	2.86E−05
ENSG00000163661	PTX3	−1.01	7.71	29.11	6.85E−08	3.24E−05
ENSG00000145386	CCNA2	−1.01	6.45	29.03	7.12E−08	3.26E−05
ENSG00000142945	KIF2C	−0.95	5.36	28.79	8.07E−08	3.58E−05
ENSG00000146678	IGFBP1	−2.51	0.45	28.44	9.65E−08	4.15E−05
ENSG00000137812	KNL1	−1.09	5.81	28.26	1.06E−07	4.44E−05
ENSG00000138182	KIF20B	−1.01	5.81	28.10	1.15E−07	4.67E−05
ENSG00000080986	NDC80	−1.05	5.15	27.56	1.52E−07	5.90E−05
ENSG00000136928	GABBR2	−0.95	6.03	27.44	1.62E−07	5.90E−05
ENSG00000137804	NUSAP1	−0.95	6.08	27.46	1.60E−07	5.90E−05
ENSG00000273294	C1QTNF3-AMACR	7.05	−2.28	27.45	1.62E−07	5.90E−05
ENSG00000131747	TOP2A	−0.97	8.09	26.97	2.07E−07	7.35E−05
ENSG00000087586	AURKA	−0.86	5.67	26.90	2.14E−07	7.42E−05
ENSG00000094880	CDC23	−0.93	5.49	26.82	2.24E−07	7.43E−05
ENSG00000161888	SPC24	−1.08	4.35	26.81	2.25E−07	7.43E−05
ENSG00000066279	ASPM	−1.18	6.89	26.71	2.36E−07	7.62E−05
ENSG00000198901	PRC1	−0.91	6.85	26.11	3.23E−07	0.000102
ENSG00000088325	TPX2	−0.86	7.28	25.96	3.49E−07	0.000108
ENSG00000138778	CENPE	−1.03	6.40	25.44	4.58E−07	0.000136
ENSG00000140525	FANCI	−0.85	5.87	25.43	4.60E−07	0.000136
ENSG00000111206	FOXM1	−0.80	6.61	25.29	4.94E−07	0.000143
ENSG00000076382	SPAG5	−0.83	5.81	25.10	5.46E−07	0.000155
ENSG00000170312	CDK1	−0.95	6.03	25.05	5.59E−07	0.000156
ENSG00000161800	RACGAP1	−0.88	5.47	24.90	6.04E−07	0.000162
ENSG00000168078	PBK	−0.93	5.37	24.91	6.02E−07	0.000162
ENSG00000146072	TNFRSF21	0.88	6.42	24.77	6.45E−07	0.00017
ENSG00000134690	CDCA8	−1.05	5.25	24.59	7.11E−07	0.000184
ENSG00000148773	MKI67	−1.17	7.99	24.06	9.33E−07	0.000237
ENSG00000102575	ACP5	−3.55	2.81	24.03	9.50E−07	0.000237
ENSG00000024526	DEPDC1	−0.99	5.61	23.90	1.02E−06	0.000249
ENSG00000099937	SERPIND1	−1.85	2.87	23.80	1.07E−06	0.000258
ENSG00000276612	FP565260.2	−6.67	−2.61	23.74	1.10E−06	0.000261
ENSG00000118193	KIF14	−1.04	5.04	23.64	1.16E−06	0.000266
ENSG00000175063	UBE2C	−1.03	5.18	23.65	1.16E−06	0.000266
ENSG00000123485	HJURP	−0.92	4.98	23.46	1.28E−06	0.000286
ENSG00000157456	CCNB2	−0.84	5.61	23.41	1.31E−06	0.000286
ENSG00000163584	RPL22L1	−0.90	5.95	23.41	1.31E−06	0.000286
ENSG00000117724	CENPF	−0.96	7.44	22.28	2.35E−06	0.000506
ENSG00000100297	MCM5	−0.86	6.30	22.12	2.56E−06	0.000535
ENSG00000105357	MYH14	4.21	−0.61	22.15	2.53E−06	0.000535
ENSG00000138160	KIF11	−0.95	6.34	22.01	2.71E−06	0.000558
ENSG00000093009	CDC45	−1.00	4.55	21.97	2.77E−06	0.000562
ENSG00000185070	FLRT2	0.76	8.29	21.87	2.92E−06	0.000584
ENSG00000116774	OLFML3	1.52	2.30	21.78	3.06E−06	0.000603
ENSG00000123689	G0S2	−1.97	2.29	21.72	3.15E−06	0.000613
ENSG00000167900	TK1	−0.76	5.68	21.56	3.43E−06	0.000655
ENSG00000184661	CDCA2	−0.93	4.87	21.54	3.46E−06	0.000655
ENSG00000013810	TACC3	−0.76	6.36	21.52	3.50E−06	0.000655
ENSG00000101057	MYBL2	−0.78	6.00	21.42	3.70E−06	0.000682
ENSG00000112742	TTK	−1.03	5.14	21.29	3.95E−06	0.000719
ENSG00000163808	KIF15	−1.04	4.74	21.00	4.59E−06	0.000826
ENSG00000011426	ANLN	−1.01	7.23	20.77	5.19E−06	0.00091
ENSG00000265107	GJA5	−6.45	−2.77	20.77	5.19E−06	0.00091
ENSG00000121152	NCAPH	−0.96	4.79	20.73	5.28E−06	0.000915
ENSG00000114631	PODXL2	1.26	3.20	20.67	5.44E−06	0.000932
ENSG00000129173	E2F8	−1.04	4.57	20.50	5.97E−06	0.00101
ENSG00000101335	MYL9	0.82	5.36	20.46	6.10E−06	0.00102
ENSG00000164109	MAD2L1	−0.92	5.54	20.32	6.55E−06	0.001082
ENSG00000104738	MCM4	−0.92	6.63	20.27	6.74E−06	0.0011
ENSG00000169679	BUB1	−0.99	5.93	20.11	7.32E−06	0.001182
ENSG00000073111	MCM2	−0.82	5.76	19.94	7.98E−06	0.00126
ENSG00000165480	SKA3	−0.99	4.57	19.95	7.95E−06	0.00126
ENSG00000075218	GTSE1	−0.89	5.40	19.81	8.54E−06	0.001333
ENSG00000267618	AC004223.3	6.30	−2.86	19.78	8.70E−06	0.001344
ENSG00000163131	CTSS	1.44	3.29	19.60	9.54E−06	0.001458
ENSG00000089685	BIRC5	−0.77	6.29	19.52	9.98E−06	0.001508
ENSG00000178999	AURKB	−0.98	4.85	19.36	1.08E−05	0.001618
ENSG00000186193	SAPCD2	−0.87	4.80	19.32	1.10E−05	0.001632
ENSG00000071539	TRIP13	−0.84	5.48	19.22	1.17E−05	0.001707
ENSG00000156504	FAM122B	−0.84	4.71	19.18	1.19E−05	0.001722
ENSG00000165092	ALDH1A1	0.69	6.16	19.14	1.21E−05	0.001741
ENSG00000123975	CKS2	−0.80	5.71	19.02	1.29E−05	0.001764
ENSG00000135476	ESPL1	−0.97	4.76	19.09	1.25E−05	0.001764
ENSG00000173597	SULT1B1	−0.97	7.01	19.02	1.29E−05	0.001764
ENSG00000189431	RASSF10	−3.28	−1.94	19.03	1.29E−05	0.001764
ENSG00000237649	KIFC1	−0.93	5.31	19.05	1.27E−05	0.001764
ENSG00000122966	CIT	−0.81	5.46	18.93	1.36E−05	0.001836
ENSG00000117650	NEK2	−0.85	4.48	18.90	1.38E−05	0.001846
ENSG00000100526	CDKN3	−0.93	4.10	18.77	1.47E−05	0.001954
ENSG00000156970	BUB1B	−0.93	5.84	18.68	1.55E−05	0.002039
ENSG00000284041	AC073111.3	−6.19	−2.96	18.66	1.57E−05	0.002041
ENSG00000115163	CENPA	−1.06	3.87	18.25	1.94E−05	0.002507
ENSG00000269891	ARHGAP19-SLIT1	6.14	−2.97	18.05	2.15E−05	0.002751
ENSG00000065328	MCM10	−1.01	4.18	17.99	2.22E−05	0.002822
ENSG00000125378	BMP4	0.74	6.11	17.97	2.25E−05	0.002823
ENSG00000079616	KIF22	−0.75	5.65	17.89	2.35E−05	0.002915
ENSG00000151640	DPYSL4	0.98	4.09	17.87	2.36E−05	0.002915
ENSG00000053747	LAMA3	−0.87	4.23	17.67	2.62E−05	0.003214
ENSG00000154175	ABI3BP	−0.84	5.18	17.65	2.65E−05	0.003219
ENSG00000183856	IQGAP3	−0.81	5.10	17.63	2.68E−05	0.003226
ENSG00000198826	ARHGAP11A	−0.92	6.08	17.48	2.91E−05	0.003475
ENSG00000228716	DHFR	−0.74	6.16	17.44	2.97E−05	0.003512
ENSG00000076003	MCM6	−0.84	6.10	17.39	3.05E−05	0.003576
ENSG00000173166	RAPH1	−0.94	5.58	17.28	3.22E−05	0.003754
ENSG00000123473	STIL	−0.90	4.92	17.23	3.31E−05	0.003819
ENSG00000117595	IRF6	1.52	3.34	17.10	3.55E−05	0.004062
ENSG00000146918	NCAPG2	−0.77	5.94	17.06	3.63E−05	0.004089
ENSG00000164647	STEAP1	−0.83	5.07	17.07	3.61E−05	0.004089
ENSG00000196878	LAMB3	−0.85	4.47	17.03	3.67E−05	0.004108
ENSG00000171241	SHCBP1	−0.86	5.58	17.01	3.72E−05	0.004126
ENSG00000150630	VEGFC	−0.96	4.78	16.89	3.95E−05	0.004353
ENSG00000090889	KIF4A	−0.77	5.75	16.62	4.56E−05	0.004974
ENSG00000121621	KIF18A	−0.92	4.27	16.61	4.59E−05	0.004974
ENSG00000101447	FAM83D	−0.88	5.40	16.54	4.77E−05	0.005136
ENSG00000169604	ANTXR1	1.03	4.62	16.47	4.93E−05	0.005267
ENSG00000096060	FKBP5	−0.68	6.90	16.31	5.38E−05	0.005672
ENSG00000167434	CA4	−4.19	−2.69	16.31	5.39E−05	0.005672
ENSG00000163092	XIRP2	1.93	1.44	16.29	5.43E−05	0.005675
ENSG00000104147	OIP5	−1.14	2.91	16.25	5.56E−05	0.005762
ENSG00000119403	PHF19	−0.64	6.01	16.19	5.74E−05	0.005868
ENSG00000211448	DIO2	1.64	0.55	16.20	5.71E−05	0.005868
ENSG00000185432	METTL7A	−0.90	5.20	16.11	5.99E−05	0.006078
ENSG00000105499	PLA2G4C	1.03	3.47	16.09	6.05E−05	0.006091
ENSG00000139734	DIAPH3	−0.80	5.47	16.01	6.30E−05	0.0063
ENSG00000163453	IGFBP7	0.68	7.65	15.92	6.60E−05	0.006561
ENSG00000140416	TPM1	0.64	8.27	15.87	6.77E−05	0.006677
ENSG00000109805	NCAPG	−0.77	6.26	15.73	7.30E−05	0.007153
ENSG00000137807	KIF23	−0.78	5.95	15.66	7.59E−05	0.007335
ENSG00000179195	ZNF664	−0.88	6.90	15.66	7.58E−05	0.007335
ENSG00000144554	FANCD2	−0.87	4.59	15.57	7.95E−05	0.007632
ENSG00000101003	GINS1	−0.83	4.79	15.51	8.19E−05	0.007811
ENSG00000137310	TCF19	−0.81	4.92	15.35	8.92E−05	0.008445
ENSG00000115008	IL1A	1.97	0.90	15.32	9.06E−05	0.008525
ENSG00000163751	CPA3	1.34	1.70	15.30	9.18E−05	0.008576
ENSG00000197632	SERPINB2	−1.60	3.06	15.21	9.61E−05	0.008924
ENSG00000133119	RFC3	−0.80	4.52	15.16	9.87E−05	0.009103
ENSG00000140545	MFGE8	0.73	5.90	15.12	0.00010086	0.009244
ENSG00000101188	NTSR1	−1.16	2.79	15.08	0.00010312	0.009391
ENSG00000171848	RRM2	−1.18	6.87	15.06	0.00010394	0.009404
ENSG00000185480	PARPBP	−0.79	4.44	15.05	0.00010467	0.009411
ENSG00000140675	SLC5A2	4.89	−2.99	14.82	0.00011808	0.010484
ENSG00000151725	CENPU	−0.74	4.67	14.83	0.00011777	0.010484
ENSG00000168243	GNG4	2.60	−1.74	14.78	0.00012062	0.010643
ENSG00000173207	CKS1B	−0.73	5.38	14.77	0.0001214	0.010646
ENSG00000010292	NCAPD2	−0.62	7.04	14.64	0.00012988	0.011319
ENSG00000173281	PPP1R3B	0.64	6.98	14.61	0.0001319	0.011425
ENSG00000164611	PTTG1	−0.64	5.60	14.58	0.00013445	0.011569
ENSG00000167261	DPEP2	2.79	−2.01	14.57	0.00013518	0.011569
ENSG00000184445	KNTC1	−0.70	5.37	14.55	0.00013641	0.011604
ENSG00000012048	BRCA1	−0.90	4.77	14.49	0.00014056	0.011791
ENSG00000092853	CLSPN	−0.87	4.95	14.49	0.0001411	0.011791
ENSG00000203668	CHML	−0.70	5.13	14.49	0.00014087	0.011791
ENSG00000101868	POLA1	−0.84	4.68	14.45	0.00014375	0.011893
ENSG00000122694	GLIPR2	0.70	5.12	14.45	0.000144	0.011893
ENSG00000149591	TAGLN	1.24	1.91	14.37	0.00015051	0.012359
ENSG00000280537	AC068946.1	2.14	−1.15	14.35	0.00015184	0.012397
ENSG00000105011	ASF1B	−0.91	4.72	14.28	0.00015721	0.012762
ENSG00000105889	STEAP1B	−0.83	4.64	14.20	0.00016442	0.013271
ENSG00000124721	DNAH8	1.61	3.82	14.17	0.00016717	0.013341
ENSG00000130816	DNMT1	−0.60	7.04	14.17	0.00016671	0.013341
ENSG00000162645	GBP2	0.60	6.15	14.14	0.00016943	0.013447
ENSG00000058804	NDC1	−0.71	5.97	14.03	0.00018026	0.014227
ENSG00000145604	SKP2	−0.67	5.33	14.00	0.0001825	0.014324
ENSG00000123219	CENPK	−0.71	4.81	13.98	0.00018513	0.014341
ENSG00000136824	SMC2	−0.81	6.08	13.97	0.00018575	0.014341
ENSG00000188517	COL25A1	2.38	−1.61	13.97	0.00018564	0.014341
ENSG00000107984	DKK1	−0.63	5.85	13.95	0.00018762	0.01437
ENSG00000204262	COL5A2	0.61	8.73	13.95	0.00018814	0.01437
ENSG00000162063	CCNF	−0.67	5.31	13.89	0.00019352	0.014701
ENSG00000258064	AC073612.1	−6.56	−2.69	13.76	0.00020816	0.01573
ENSG00000120802	TMPO	−0.66	7.44	13.64	0.00022161	0.016569
ENSG00000163507	CIP2A	−0.82	5.14	13.64	0.0002212	0.016569
ENSG00000186185	KIF18B	−0.86	4.35	13.61	0.00022501	0.016735
ENSG00000149503	INCENP	−0.86	5.22	13.41	0.00025051	0.018535
ENSG00000167601	AXL	−0.55	7.33	13.28	0.00026806	0.019731
ENSG00000139618	BRCA2	−0.91	4.49	13.19	0.00028185	0.020639
ENSG00000163554	SPTA1	2.18	−0.67	13.16	0.00028663	0.020881
ENSG00000100311	PDGFB	0.82	6.70	13.08	0.00029782	0.021586
ENSG00000106069	CHN2	0.94	3.27	12.98	0.00031547	0.022542
ENSG00000239389	PCDHA13	6.39	−2.80	12.99	0.00031302	0.022542
ENSG00000258947	TUBB3	0.59	7.10	12.97	0.00031577	0.022542
ENSG00000144354	CDCA7	−0.74	4.99	12.94	0.0003212	0.022815
ENSG00000123080	CDKN2C	−0.81	4.00	12.93	0.00032414	0.022909
ENSG00000103489	XYLT1	1.13	2.69	12.83	0.00034094	0.023859
ENSG00000178538	CA8	−1.71	0.71	12.83	0.00034088	0.023859
ENSG00000163535	SGO2	−0.80	5.30	12.81	0.00034481	0.024012
ENSG00000133110	POSTN	−1.11	3.05	12.78	0.00035075	0.024306
ENSG00000035499	DEPDC1B	−0.85	4.24	12.75	0.00035616	0.024325
ENSG00000150540	HNMT	0.80	4.05	12.75	0.0003557	0.024325
ENSG00000176890	TYMS	−0.61	6.99	12.75	0.00035522	0.024325
ENSG00000261459	AC002310.5	−6.20	−2.95	12.71	0.00036311	0.024681
ENSG00000117593	DARS2	−0.67	5.54	12.68	0.00036955	0.024999
ENSG00000111341	MGP	−0.57	6.67	12.65	0.00037599	0.025314
ENSG00000058085	LAMC2	0.75	4.73	12.57	0.00039222	0.026282
ENSG00000103257	SLC7A5	−0.71	4.74	12.49	0.00040895	0.027275
ENSG00000214357	NEURL1B	−1.41	0.86	12.46	0.0004151	0.027556
ENSG00000258555	SPECC1L-ADORA2A	6.16	−2.96	12.37	0.00043612	0.028683
ENSG00000268643	AC006486.1	−6.10	−3.02	12.37	0.00043584	0.028683
ENSG00000186871	ERCC6L	−0.97	3.80	12.31	0.00045156	0.029562
ENSG00000128944	KNSTRN	−0.64	5.27	12.26	0.00046273	0.029611
ENSG00000134222	PSRC1	−0.89	3.87	12.28	0.00045803	0.029611
ENSG00000142731	PLK4	−0.89	4.91	12.27	0.00046122	0.029611
ENSG00000147536	GINS4	−0.85	4.14	12.28	0.00045741	0.029611
ENSG00000267022	AC067968.1	−2.73	−1.12	12.26	0.00046177	0.029611
ENSG00000155093	PTPRN2	1.40	0.73	12.23	0.00047029	0.029846
ENSG00000188229	TUBB4B	−0.69	8.39	12.23	0.00047061	0.029846
ENSG00000215252	GOLGA8B	0.68	4.46	12.21	0.00047536	0.030013
ENSG00000149573	MPZL2	−0.67	5.40	12.20	0.00047877	0.030095
ENSG00000129195	PIMREG	−0.79	4.07	12.18	0.00048388	0.030282
ENSG00000152104	PTPN14	−0.60	7.17	12.16	0.00048777	0.030391
ENSG00000197457	STMN3	2.81	3.31	12.14	0.00049373	0.030629
ENSG00000168874	ATOH8	0.84	3.70	12.13	0.00049593	0.030631
ENSG00000255073	ZFP91-CNTF	−5.43	−3.45	12.10	0.00050547	0.031085
ENSG00000188486	H2AFX	−0.63	6.27	12.02	0.00052646	0.032209
ENSG00000213297	ZNF625-ZNF20	1.41	0.93	12.01	0.00052828	0.032209
ENSG00000102007	PLP2	−0.55	6.27	11.97	0.00053967	0.032496
ENSG00000135842	FAM129A	−1.02	3.29	11.97	0.00053985	0.032496
ENSG00000168843	FSTL5	1.09	3.04	11.98	0.00053834	0.032496
ENSG00000178878	APOLD1	−0.89	3.58	11.94	0.00055082	0.033017
ENSG00000113368	LMNB1	−0.66	6.70	11.92	0.00055512	0.033135
ENSG00000137473	TTC29	1.72	−0.20	11.88	0.0005664	0.033666
ENSG00000148848	ADAM12	1.25	1.79	11.86	0.00057272	0.0339
ENSG00000114346	ECT2	−0.68	5.97	11.84	0.00058063	0.034226
ENSG00000196739	COL27A1	0.57	5.97	11.77	0.00060245	0.03522
ENSG00000258677	AC022826.2	−1.79	−0.91	11.77	0.00060175	0.03522
ENSG00000129810	SGO1	−0.89	3.52	11.75	0.00060846	0.035425
ENSG00000198830	HMGN2	−0.53	8.42	11.73	0.00061337	0.035566
ENSG00000174371	EXO1	−0.93	3.73	11.69	0.00062871	0.036307
ENSG00000127564	PKMYT1	−0.90	3.52	11.66	0.00063706	0.03664
ENSG00000100368	CSF2RB	0.55	5.77	11.64	0.00064394	0.036738
ENSG00000135451	TROAP	−0.73	4.10	11.64	0.00064385	0.036738
ENSG00000092470	WDR76	−0.72	4.40	11.60	0.00065967	0.037485
ENSG00000143401	ANP32E	−0.56	6.78	11.58	0.00066731	0.037768
ENSG00000257411	AC034102.2	1.93	0.75	11.54	0.0006809	0.038384
ENSG00000146670	CDCA5	−0.72	5.02	11.50	0.00069768	0.039175
ENSG00000167325	RRM1	−0.57	6.89	11.47	0.00070846	0.039623
ENSG00000152253	SPC25	−0.89	3.82	11.46	0.00071221	0.039677
ENSG00000122952	ZWINT	−0.71	5.53	11.43	0.00072353	0.04015
ENSG00000121966	CXCR4	1.02	5.44	11.40	0.00073256	0.040493
ENSG00000101224	CDC25B	−0.56	6.33	11.38	0.00074161	0.040707
ENSG00000136490	LIMD2	0.60	5.23	11.38	0.00074216	0.040707
ENSG00000127528	KLF2	−0.82	4.46	11.35	0.00075267	0.041125
ENSG00000151388	ADAMTS12	0.85	3.45	11.34	0.00075879	0.041163
ENSG00000154839	SKA1	−0.87	4.20	11.34	0.00075917	0.041163
ENSG00000119969	HELLS	−0.65	4.77	11.30	0.00077592	0.041911
ENSG00000108055	SMC3	−0.53	6.68	11.28	0.00078172	0.042065
ENSG00000125695	AC046185.1	5.80	−3.20	11.24	0.00079891	0.042828
ENSG00000146411	SLC2A12	1.26	2.09	11.23	0.00080506	0.042888
ENSG00000159167	STC1	−0.93	4.54	11.23	0.00080607	0.042888
ENSG00000181634	TNFSF15	0.78	5.81	11.19	0.00082212	0.043579
ENSG00000111665	CDCA3	−0.78	3.97	11.16	0.00083518	0.044106
ENSG00000067141	NEO1	−2.49	−2.06	11.10	0.00086297	0.045237
ENSG00000099282	TSPAN15	0.66	6.21	11.10	0.00086289	0.045237
ENSG00000120256	LRP11	0.57	5.86	10.97	0.00092765	0.048365
ENSG00000145990	GFOD1	−0.59	5.47	10.96	0.00093285	0.048365
ENSG00000166250	CLMP	−1.02	2.47	10.96	0.00093028	0.048365
ENSG00000072501	SMC1A	−0.59	6.94	10.94	0.00093894	0.048504
ENSG00000112378	PERP	−0.59	7.18	10.92	0.00095053	0.048925
ENSG00000077152	UBE2T	−0.67	4.49	10.90	0.00096365	0.049243
ENSG00000198553	KCNRG	1.57	−0.55	10.90	0.00096042	0.049243
ENSG00000120337	TNFSF18	0.87	4.84	10.87	0.00097964	0.049881

Bioinformatic analyses highlighted a potential role for the Src Signaling pathway, and other pathways, in the pathology of IA. We evaluated the potential link to the Src signaling pathway below.

EXAMPLE 2: THSD1 Suppresses Podosome Formation and Function, and this Mechanism Compromises Cerebrovascular Integrity and Promotes IA Development

Intracranial aneurysm (IA) is a weakened area in the wall of cerebral artery that leads to a bulging in a brain blood vessel. The rupture of IA causes aneurysmal subarachnoid hemorrhage (SAH), a devastating form of stroke. More than 30% will die due to SAH and more than half of survivors will never return to independent living. Unfortunately, there are no treatments for IA except open or endovascular surgery. Attempts to find new therapeutic avenues are greatly hindered by the lack of knowledge of the gene(s) and pathways responsible for IA development and growth.

Podosomes are subcellular organelles located at cell-ECM interaction sites in different cell types including a variety of endothelial cells. A podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM (FIG. 3). Unlike other adhesive structures such as focal adhesion complexes that tether the endothelium to the ECM, the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9. Among all the upstream events that occur during podosome formation, the best characterized is the activation of SRC, a non-receptor tyrosine kinase. When SRC is enriched and activated in pre-podosome structures, it phosphorylates a wide array of substrates. For instance, SRC phosphorylates cortactin and promotes actin assembly in podosomes. See, e.g., FIG. 3.

The present disclosure considered the molecular pathways affected by the knock-down of THSD1 and investigated the role of podosome formation and function role in THSD1-mediated aneurysm development.

Materials and Methods

Cell Culture

HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.

Western Bot

Cells were lysed in 1% Triton lysis buffer and sonicated briefly before centrifuged at 18506 g for 30 min at 4° C. Total cell lysates were added by 2X SDS sample buffer and then subjected to discontinuous SDS-PAGE analysis. Proteins were transferred to nitro-cellulose membranes using a Bio-Rad (Hercules, Calif.) mini transfer apparatus followed by blocking with 5% nonfat milk. Primary antibodies and secondary antibodies were used usually at 1:1000 and 1:10000 dilutions respectively before using an Odyssey system to detect the fluorescence signal.

Immunofluorescence

Cells were grown on glass coverslips in the DMEM medium for 16 h before fixed with 4% formaldehyde-phosphate-buffered saline (Electronic Microscopy Sciences, 15710; Fisher Scientific, BP399-500) for 10 min. Cells were incubated with primary antibodies against focal adhesion markers such as paxillin overnight at 4° C. after 1 h blocking by 5% goat serum. Secondary antibodies conjugated with Alexa Fluor 488 or 594 were used to visualize the localization. Image was taken with a Leica Confocal Microscope and processed by ImageJ and GraphPad 7 software for statistical analysis.

Intracranial Hemorrhage in Zebrafish

Anti-sense morpholinos against thsd1, atg5 or talin was injected into one-cell stage zebrafish singly or in combination to silence the gene accordingly. At day 2 to day 3, embryos will be checked under regular optical microscope. Embryos with intracranial hemorrhages will be counted and imaged by Olympus microscope.

MicroFil Injection in Mice

Prepare MicroFil (Flow Tech, Inc. Carver, Mass.) casting solution according to the manufacturer's instruction: Mix 5 ml of MV diluent with 4 ml of filtered MV-112 compound (yellow). Add 450 μl (5%) of catalyst (MV curing agent). Use 10 ml syringe to inject MicroFil mixture into the left ventricle after the blood was flushed out by saline. Inject MicroFil mixture slowly into the left ventricle at approximately 3 ml/min.

En Face Immunostaining of Circle of Willis in Mice

For en face immunostaining, isolated intracerebral arteries were dissected out from the brain with micro-needle and micro-scissors under the stereomicroscope. For immunofluorescent staining, tissue samples were washed in phosphate buffered saline (PBS), blocked with 5% goat serum in PBST (0.1% Triton-X in PBS), then incubated with primary antibodies followed by the appropriate fluorescent-labeled secondary antibodies.

Loss-of-Function of THSD1 Induces Endothelial Podosome Formation and Function.

Formation:

FIGS. 4a-4b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation in THSD1-deficient HBMECs. FIG. 4a illustrates podosomes such as puncta (arrowheads) or rosette (arrow) were assessed by co-immunostaining of cortactin and actin in control or THSD1-deficient HBMECs. FIG. 4b illustrates the percentage in HBMECs (n=3 independent experiments) and student t-test was used for statistical difference. *p<0.05, Scale bar: 10 μm.

Function:

FIGS. 5a-5b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function. FIG. 5a shows representative images for Alexa 488-conjugated Collagen-IV (Co-IV) degradation in control or THSD1-deficient HBMECs. FIG. 5b shows a tabulation of the degradation index (degraded area per cellular profile) calculated from at least 9 different fields (10X objective) in HBMECs (n=3 independent experiments), and student t-test was used for statistical difference. *p<0.05, Scale bar: 10 μm.

Loss-of-Function of THSD1 Increase MT1-MMP Cell Surface Exposure.

FIGS. 6a-6b are experimental results depicting that loss-of-function of THSD1 increases MT1-MMP cell surface exposure. FIG. 6a shows a Western Blot illustrating that biotinylated cell-surface proteins were pull down by streptavidin beads from control and THSD1-deficient HBMECs. The level of MT1-MMP were revealed by Western blot and quantified in FIG. 6b. TCL: total cell lysates; GAPDH as loading control; **p<0.01 by student t-test.

FIGS. 7a-7b are experimental results depicting that MMP2 is activated upon THSD1 knockdown in HBMECs. FIG. 7a illustrates cell extracts and supernatants from control and THSD1-deficient HBMECs were analyzed by gelatin zymography. Loading was normalized by total protein concentration. Quantification was calculated by a fold change of active MMP2 expression. See FIG. 7b. ***p<0.001 by student t-test.

EXAMPLE 3: In-Vivo SRC Inhibition Rescues Cerebrovascular Integrity Defects

In-Vivo Zebrafish Model

Intracranial aneurysm and hemorrhage are tightly associated with compromised cerebrovascular integrity. Recently, zebrafish have gained additional popularity as a vertebrate model organism for studying the cerebrovasculature. Since zebrafish embryos are transparent, intracranial hemorrhage can be directly observed using a standard microscope. Furthermore, zebrafish fecundity and rapid development permits rapid phenotypic evaluation as intracranial hemorrhage in zebrafish fry are detectable as early as 2-3 days post fertilization. Importantly, gain-of-function and loss-of-function approaches are well established in zebrafish that include morpholino and mRNA injections and more recently, through applications of CRISPR/Cas9 technology.

FIGS. 8a-8b are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish, and treatment with a PP2 SRC inhibitor prevents intracranial hemorrhage in Thsd1-deficient zebrafish. FIG. 8a is a picture illustrating a prominent cerebral hemorrhage phenotype was observed in thsd1 knockdown zebrafish. FIG. 8b is a chart quantifying the number of embryos with intracranial hemorrhage (%). As shown in FIG. 8b PP2 treatment (SRC inhibitor) ameliorated Thsd1-dependent hemorrhage incidents, while DMSO treatment (control) did not (comparing grey bars in b). n>120 embryos were analyzed. *p<0.05.

FIG. 9 (FIG. 9) are experimental results depicting that loss-of-function of Thsd1 promotes IA formation in circle of Willis in mice. IAs were visualized by Microfil including a PCA (top panel), a left Pcom (middle panel), and a right ICA (bottom panel) as indicated by arrows. n=10

FIGS. 10a-10b are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice. Podosomes were revealed by en face immunostaining against cortactin (green) and VE-cadherin (red) in CoW of Thsd1+/+(WT, n=9) vs. Thsd1−/− (KO, n=10) mice. Student t-test was used for statistical difference. **p<0.01. Scale bar: 10 μM. FIG. 10a illustrates representative images of the staining and FIG. 10b is a chart representation quantitation of the same.

EXAMPLE 4: Genetic Engineering of Non-Human Animal Models Encoding Distinct THSD1 Rare Variants for In-Vivo Assessment of Efficacy of Various Autophagy Inhibitors

Identified Conserved Sequences Suitable for Assessing Cross Species Efficacies of Treatments:

A protein alignment between human and zebrafish THSD1 was performed. See FIG. 11 illustrates the result of the alignment with black color indicating identical amino acids and the grey color indicating similar amino acids between humans and zebra fish. The alignment underscored that three rare variants of THSD1 identified in the aforementioned examples were conserved between human and zebrafish. The conserved amino acids include R450, R460 and T653. See, e.g., FIG. 12. The conserved amino acids were used as a rational for generating animal models of the human condition. The following constructs were generated:

TABLE 5
1 pCS2 + zf-thsd1-WT-CT2FLAG (for full rescue, positive control)
2 pCS2 + zf-thsd1-R449X-CT2FLAG (corresponding to R450X
  in human)
3 pCS2 + zf-thsd1-R459W-CT2FLAG (corresponding to R460W
  in human)
4 pCS2 + zf-thsd1-T665I-CT2FLAG (corresponding to T653I
  in human)

Mutations R449X, R459W and T6651 were found to be conserved between human and zebrafish. Subsequently, the nucleic acid constructs encoding these sequences were introduced into zebrafish thsd1 by PCR mutagenesis. Specifically, the thsd1 variant or the WT sequence was subcloned into pCS2+ zebrafish vector for expression. These constructs were used as tools to study cerebrovascular integrity mediated by thsd1 WT and rare variants in the zebrafish animal model selected to model a human condition.

In-Vivo Zebrafish Model

To test the hypothesis that podosome modulation via SRC inhibition could treat intracranial hemorrhage in-vivo 80-120 different zebrafish animals genetically engineered as described in Table 5 were treated with three distinct SRC inhibitors, namely, PP2, SU5565, or dasatinib. Control (DMSO) or other known SRC inhibitors including PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) were added to embryo water at 1 dpf for 24 hours. All embryos were injected by thsd1-MO and the hemorrhagic incidence was counted under microscope. For each injection, around 80-120 embryos were injected. *p<0.05, **p<0.01. See FIG. 13.

The data demonstrates a body of work that first identified and studied a large number of human families with a disease. After significant effort in contacting individuals and sequencing the families shown in FIG. 1A, the present disclosure first identified THSD1 as a gene associated with sub-cranial hemorrhage. In specific cases, a single codon substitution was identified as responsible for the phenotype LSF, R460W, E466G, G600E, P639L, T6531, or S775P. The inventors developed a plethora of nucleic acid constructs, cell lines, and transgenic animal models to study the condition. Subsequently, a detailed characterization of molecular pathways whose transcription was disturbed in the disease condition was used to identify potential druggable targets and pathways for treatment. One such pathway was podosome formation and function. Three distinct SRC inhibitors were tested including PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and its ability to treat subjects, particularly its ability to treat subjects susceptible to an aneurism rupture was tested by counting the hemorrhagic incidence under the microscope on an animal model. The results illustrate that podosome modulation provides a treatment for modulating the rupture of an aneurysm in vivo.

EXAMPLE 6: Kits

The disclosure contemplates kits comprising podosome modulators such as the podosome modulators selected from the group consisting of PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and instructions for use of the same in the treatment of a condition disclosure herein. Specifically, the disclosure contemplates use of these drugs in treating an intracranial aneurysm.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, 6.

Claims

1. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation.

2. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome function.

3. The method of any one of claim 1 or 2, wherein the compound is a Src inhibitor.

4. The method of claim 3, wherein the Src inhibitor is PP2.

5. The method of any one of claim 1 or 2, wherein the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.

6. The method of claim 5, wherein the compound is SU6656.

7. The method of claim 5, wherein the compound is KX2-391.

8. The method of claim 5, wherein the compound is saracatinib.

9. The method of claim 5, wherein the compound is bosutinib.

10. The method of claim 5, wherein the compound is dasatinib.

11. The method of claim 5, wherein the compound is ponatinib.

12. The method of any one of claims 1-5, wherein the aneurysm is an intracranial aneurysm.

13. The method of any one of claims 1-5, wherein the aneurysm is an aortic aneurysm.

14. The method of any one of claims 1-13, wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

15. The method of claim 14, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

16. The method of claim 14, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.

17. The method of claim 14, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.

18. The method of claim 14, wherein the single codon substitution in the at least one THSD1 allele is at codon LSF, R460W, E466G, G600E, P639L, T6531, S775P.

19. The method of claim 1, wherein the compound that modulates podosome formation or function is administered systemically.

20. The method of claim 19, wherein systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal.

21. The method of claim 1, wherein the compound that modulates podosome formation or function is administered locally.

22. The method of claim 21, wherein local administration includes, but is not limited to, (i) intracranial; (ii) intra-ocular; (iii) intra-nasal; (iv) intrathecal (v) vescular.

23. The method of claim 1, wherein the subject is a human.