Biomarkers for Thoracic Aortic Aneurysm

Abstract

A method for detecting thoracic aortic aneurysm (TAA) or susceptibility to TAA in a subject, comprising measuring the amount of tetrahydrobiopterin (H4B) present in a test sample; and comparing the measured amount of H4B with a standard amount of H4B. A decrease in the amount of H4B present in the test sample as compared to the standard indicates TAA or susceptibility to TAA. The method may further comprise identifying candidates for further testing or monitoring of TAA, e.g., by ultrasound or by repeating the test for H4B after one or more specified time intervals. Additionally, the method may further comprise prescribing treatment for TAA to the subject, such as folic acid therapy and / or DHFR therapy, including gene therapy, and other therapies effective for re-coupling eNOS and / or therapies targeting uncoupled eNOS. Methods for monitoring the therapeutic efficacy of TAA treatment and for assessing the severity of TAA or TAA risk are also described.

Description

[0001] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. HL077440 awarded by the National Institutes of Health. The government has certain rights in this invention. FIELD OF THE INVENTION

[0003] This invention generally relates to a novel biomarker for thoracic aortic aneurysm, and methods for diagnosing, risk detecting, and monitoring disease progression and response to aneurysm treatment using such a biomarker. BACKGROUND OF THE INVENTION

[0005] Thoracic aortic aneurysm (TAA) is a prevalent human disease affecting 4.2% of the general population (Booher AM et al., Am Heart J. 2011). Despite surgical correction, there are no effective oral therapies or biomarkers for early detection, or for monitoring treatment efficacy and postoperative recurrence.

[0006] There remains a need for biomarkers for TAA. In particular, there remains a need for biomarkers that can be used to screen for, detect, and monitor TAA and to identify those individuals predisposed to developing TAA. SUMMARY OF THE INVENTION

[0007] The present invention provides a method for detecting thoracic aortic aneurysm (TAA) or susceptibility to TAA in a subject. In some embodiments, the method comprises (a) measuring the amount of tetrahydrobiopterin (H4B) present in a test sample; and (b) comparing the measured amount of H4B present in the test sample to a standard amount of H4B. In some embodiments, the method comprises (a) contacting a test sample from a subject with an assay device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; and (b) comparing the measured amount of H4B present in the test sample to a standard amount of H4B. In exemplary embodiments of the invention, the sample comprises serum, plasma, or whole blood. A decrease in the amount of H4B present in the test sample as compared to the standard indicates TAA or susceptibility to TAA. In some embodiments, a statistically significant decrease in the amount of H4B present in the test sample as compared to the standard indicates TAA or susceptibility to TAA. In other embodiments, the decrease in H4B is at least about 10% less than the standard, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80% or 90% less. A smaller decrease generally indicates susceptibility to TAA, while a larger decrease is more likely to indicate the presence of TAA.

[0008] Accordingly, the method may further comprise identifying a subject as a candidate for further testing or monitoring of TAA, e.g., by ultrasound or by repeating the test for H4B after one or more specified time intervals. Additionally, the method may further comprise prescribing TAA treatment to a subject with a reduction in H4B compared to a standard. Examples of treatment include folic acid therapy and / or DHFR (dihydrofolate reductase) therapy, including gene therapy.

[0009] There is also provided a method for monitoring the therapeutic efficacy of TAA in a subject. In one embodiment, the method comprises (a) measuring the amount of H4B present in a first test sample obtained from the subject at a first time point; (b) measuring the amount of H4B present in a second test sample obtained from the subject at a second time point; and (c) comparing the measured amounts of H4B present in the first test sample and the second test sample. In one embodiment, the method comprises (a) contacting a first test sample obtained from the subject at a first time point with an assay device capable of measuring the amount of H4B present in the test sample; (b) contacting a second test sample obtained from the subject at a second time point with an assay device capable of measuring the amount of H4B present in the test sample; and (c) comparing the measured amounts of H4B present in the first test sample and the second test sample.

[0010] A treatment is administered to the subject prior to the second time point, and an increase in the amount of H4B present in the second test sample as compared to the first test sample indicates an effective treatment of TAA. This method can be initiated at the start of treatment or after a treatment regimen has been executed. In some embodiments, a statistically significant increase in the amount of H4B present in the second sample as compared to the first sample indicates an effective treatment of TAA. In other embodiments, the increase in H4B is at least about 10% greater than the first sample, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80%, 90%, 100%, 150%, 200% or more. The method optionally further comprises prescribing a modified TAA treatment to a subject with a decrease or increase in H4B as compared to the first sample or to a standard.

[0011] In addition, the present invention provides a method for assessing the severity of thoracic aortic aneurysm (TAA) or the risk of TAA in a subject. In one embodiment, the method comprises (a) optionally, contacting a test sample from the subject with an assay device capable of measuring the amount of H4B present in the test sample; (b) measuring the amount of H4B present in the test sample; and (c) comparing the measured amount of H4B present in the test sample with the measured amount of H4B present in a standard. The degree of reduction in the amount of H4B present in the test sample relative to the standard indicates the severity or risk of TAA in the subject. In some embodiments, a statistically significant reduction in the amount of H4B present in the test sample relative to the standard indicates TAA or susceptibility to TAA. In other embodiments, the reduction in H4B is at least about 10% less than the standard, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80% or 90% less. A smaller reduction generally indicates susceptibility to TAA or a milder case of TAA, while a larger reduction is more likely to indicate the presence of TAA or a more severe case of TAA. Depending on the amount of H4B present in the test sample, the subject can be monitored or treated as described herein.

[0012] In exemplary embodiments, the assay device comprises a high performance liquid chromatography (HPLC) column, or an immunoassay kit, such as an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescent assay kit or other conventional assay kits. Accordingly, the present invention further provides a kit comprising a reagent and / or an assay device for detecting H4B. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 . Reduced levels of tetrahydrobiopterin (H4B) can be used as a marker for TAA formation. A significant decrease in the levels of circulating biomarkers was found in patients with TAA.

[0014] Figure 2 . Fbn1 C1039G / + Genotyping of mice. WT: wild type (164 bp, Fbn1 + / + ), Het: heterozygous (212 bp and 164 bp, Fbn1 C1039G / + ).

[0015] Figures 3A - 3B . Dietary folic acid-mediated eNOS recoupling abolished the dilation of the aortic root and abdominal aorta in Fbn1C1039G / + animals. The diameters of the aortic root (3A) and abdominal aorta (3B) were measured using echocardiography and were found to be significantly increased in Fbn1C1039G / + mice, and this increase was greatly attenuated by dietary folic acid-mediated eNOS recoupling. Data are presented as mean ± SEM, n = 19 - 38.

[0016] Figures 4A - 4B .Folic acid diet prevents eNOS uncoupling and preserves Fbn1 C1039G / + NO bioavailability in mice. (4A) Total superoxide production from aortic homogenates was determined by electron spin resonance (ESR) in the presence or absence of L-NAME (NOS inhibitor). At 8 and 12 weeks, Fbn1 C1039G / + eNOS uncoupling activity (L-NAME-inhibitable superoxide production) in mice was completely attenuated by folic acid diet. n = 5 - 7. (4B) NO bioavailability in aortic tissues was determined by ESR. At 8 and 12 weeks, folic acid diet significantly preserved Fbn1 C1039G / + NO bioavailability in mice. n = 7 - 12. Data are presented as mean ± SEM, *p <= 0.05, **p <= 0.01.

[0017] Figures 5A - 5E .Folic acid diet increases Fbn1 + / + and Fbn1 C1039G / + tissue and circulating H4B levels in animals. Aortic H4B levels (5A) and plasma H4B levels (5B) were determined by HPLC (n = 5 - 7). Folic acid diet significantly increased wild-type Fbn1 + / + littermates and Fbn1 C1039G / + tissue and circulating H4B levels in mice. (5C) Correlation analysis of aortic H4B and circulating H4B levels indicated that circulating H4B accurately reflected tissue H4B levels, n = 35. (5D) Correlation between aortic root diameter and aortic H4B levels indicated that lower tissue H4B levels were associated with greater dilation of the aortic root, n = 35. (5E) Correlation between aortic root diameter and plasma H4B levels indicated that lower circulating H4B levels were associated with greater dilation of the aortic root, n = 35. Data are presented as mean ± SEM.

[0018] Figures 6A - 6E .Folic acid diet prevents eNOS uncoupling by upregulating DHFR protein expression and activity in Fbn1c1039 / + mice. Endothelial cells (ECs) were isolated from the aortas of Fbn1 + / + and Fbn1 c1039G / + mice. (6A) Using actin as an internal control, the expression levels of DHFR and eNOS were examined by Western blotting. (6B) Densitometric quantification of DHFR expression. (6C) Densitometric quantification of eNOS expression. DHFR activity was determined by HPLC in isolated ECs (6D) and denuded aortas (6E). Data are presented as mean ± SEM, n = 5 - 8.

[0019] Figures 7A - 7F .Fbn1 c1039G / + Protein expression levels of GTPCHI, TGFβ, and NOX4 in animals. (7A) Compared with Fbn1 + / + mice, representative western blot of GTPCHI protein expression in Fbn1 c1039G / + mice. (7B) Densitometric quantification of GTPCHI protein expression, indicating downregulated GTPCHI in Fbn1 c1039G / + mice. (7C) Compared with Fbn1 + / + mice, representative western blot of inactive and mature TGFβ proteins in the aorta of Fbn1 c1039G / + mice. (7D) Densitometric quantification of inactive and mature TGFβ expression, indicating upregulated mature TGFβ protein in Fbn1 c1039G / + mice. (7E) Compared with Fbn1 + / + mice, representative western blot of NOX4 protein expression in Fbn1 c1039G / + mice. (7F) Densitometric quantification of NOX4 expression, indicating upregulated NOX4 protein expression in Fbn1 c1039G / + mice. Data are presented as mean ± SEM, n = 4 - 9. *p < 0.05.

[0020] Figures 8A - 8E .TGFβ blocking antibody attenuates aortic root dilation through NOX4 inhibition-dependent eNOS recoupling in Fbn1 C1039G / + mice. (8A) Representative western blot of NOX4 expression in the aorta of Fbn1 C1039G / + mice treated with TGFβ blocking antibody. (8B) Densitometric quantification of NOX4 protein expression, indicating inhibition by TGFβ blocking antibody. (8C) Reduction in aortic root diameter in Fbn1 C1039G / + mice at 3 and 4 weeks after injection of TGFβ blocking antibody. (8D) No change in abdominal aortic diameter in Fbn1 C1039G / + mice up to 4 weeks after injection of TGFβ blocking antibody. (8E) Total superoxide production in aortic tissues of Fbn1 C1039G / + mice in the presence or absence of L-NAME after 4 weeks of treatment with TGFβ blocking antibody. Results indicate that TGFβ blocking antibody recouples eNOS. Data are presented as mean ± SEM, n = 4 - 9. *p < 0.05.

[0021] Figure 9Novel therapeutic effects of FA and TGFβ blocking antibodies on TAA by impairing the TGFβ / NOX4 / eNOS uncoupling axis. FA significantly impaired dilation of the aortic root and abdominal aorta via the DHFR / H4B / eNOS recoupling / NO pathway, while anti-TGFβ antibodies reduced NOX4 expression to allow Fbn1 C1039G / + eNOS in mice is recoupled, thereby impairing TAA formation. DETAILED DESCRIPTION

[0022] This article describes the unexpected discovery that plasma levels of tetrahydrobiopterin (H4B) can be used as a novel biomarker for thoracic aortic aneurysm (TAA) and that TAA formation can be abolished with folic acid (FA) treatment. This finding is surprising given the epigenetic determinism of TAA and its association with congenital disorders such as bicuspid aortic valve, Marfan syndrome, and Loeys-Dietz syndrome.

[0023] As described in U.S. Patent Publication No. 20140308686, published on October 16, 2014, circulating tetrahydrobiopterin (H4B) can accurately predict its tissue levels, which are directly involved in the pathogenesis of abdominal aortic aneurysm (AAA). Based on a large amount of data from several novel and classic animal models and human patients, H4B can be used as a biomarker for AAA. Traditionally, TAAs are considered to have more genetic certainty. Nevertheless, current data surprisingly show that genetic defects can be introduced into similar mechanistic pathways recently identified for AAA. Using two different AAA models, angiotensin II-infused hph-1 and apoE knockout mice, previous studies have demonstrated that plasma H4B levels are closely correlated with tissue H4B levels, both of which are reduced in AAA and restored by folic acid treatment.

[0024] Oral administration of folic acid allows eNOS to recouple, thereby reducing oxidative stress and increasing the bioavailability of nitric oxide, which in turn prevents vascular remodeling prior to TAA. This is due to folic acid upregulation of the eNOS cofactor salvage enzyme dihydrofolate reductase (DHFR). Therefore, subjects identified by the H4B test according to the present invention can be treated with folic acid or other therapies that promote DHFR, such as DHFR gene therapy. This early detection can reduce or eliminate the need for surgical repair and the risk of rupture.

[0025] Definition

[0026] Unless otherwise specified, all scientific and technical terms used in this application have the meanings commonly used in the art. As used in this application, the following words or phrases have the specified meanings.

[0027] As used herein, a "determination device" refers to an analytical instrument or apparatus commonly used to analyze, measure, and / or detect the presence of a chemical substance. Typical examples of such an instrument are high performance liquid chromatography (HPLC) columns. Other chromatographic instruments, as well as immunoassays, chemiluminescence assays, or other conventional detection assays, can be used. A typical example of an immunoassay is ELISA.

[0028] As is understood by those skilled in the art, a sample obtained from a subject can be contacted with an analytical instrument either directly or after first contacting it with a solvent or other preparation medium.

[0029] As used herein, a "control" sample is typically a sample obtained from one or more normal healthy subjects or, where appropriate, from the same subject but at a time when the subject is known to be in a healthy state. A recognized normal level of a reference analyte is also suitable as a control for comparison, referred to herein as a "standard".

[0030] As used herein, a "pharmaceutically acceptable carrier" or "excipient" includes any material that, when combined with an active ingredient, allows the ingredient to remain biologically active and does not react with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), and various types of wetting agents. Compositions containing such carriers are formulated by well-known methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0031] As used herein, unless otherwise clearly indicated, "a" or "an" means at least one.

[0032] As used herein, "prevent" or "preclude" a disorder or disease means to hinder, reduce, or delay the onset or progression of the disorder or disease.

[0033] Method

[0034] The present invention provides a method for detecting thoracic aortic aneurysm (TAA) or susceptibility to TAA in a subject. In some embodiments, the method comprises (a) measuring the amount of tetrahydrobiopterin (H4B) present in a test sample; and (b) comparing the measured amount of H4B present in the test sample with a standard amount of H4B. In some embodiments, the method comprises (a) contacting a test sample from a subject with an assay device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; and (b) comparing the measured amount of H4B present in the test sample with a standard amount of H4B. In some embodiments, the method comprises detecting a decrease in the amount of H4B present in the test sample compared to a standard, control, or reference value. In exemplary embodiments of the invention, the sample comprises serum, plasma, or whole blood. A decrease in the amount of H4B present in the test sample compared to a standard indicates TAA or susceptibility to TAA. The method may further comprise prescribing treatment for TAA to a subject with a decrease in H4B compared to a standard. Examples of treatment include folate therapy and / or dihydrofolate reductase (DHFR)-targeted therapy, including gene therapy, and any other pharmacological or other therapy effective to improve DHFR function, which will increase H4B levels and prevent, delay, or improve TAA. Similarly, treatment may include other countermeasures directed at re-coupling eNOS and / or therapies targeting uncoupled eNOS.

[0035] In some embodiments, a statistically significant decrease in the amount of H4B present in the test sample compared to a standard indicates TAA or susceptibility to TAA. A difference in circulating H4B levels in the range of 0.1 to 0.4 pmol / μg can be detected in people with or without TAA. In some embodiments, an H4B level dropping to about 0.20 - 0.15 pmol / μg indicates TAA, and a level below about 0.15 - 0.10 pmol / μg indicates severe TAA. In other embodiments, the decrease in H4B is at least about 10% less than the standard, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80% or 90% less. A smaller decrease, e.g., in the range up to about 0.20 pmol / μg, generally indicates susceptibility to TAA, while a larger decrease, e.g., down to less than about 0.15 pmol / μg, is more likely to indicate the presence of TAA. The amount of circulating H4B is related to the size of the aneurysm.

[0036] In some embodiments, the standard amount of H4B for reference is taken from levels that are recognized by those skilled in the art as normal for healthy subjects without TAA, and such levels will vary depending on age and gender. In other embodiments, the standard for comparison in the method is a sample obtained from normal healthy control subjects. In other embodiments, the standard for comparison is a test sample previously obtained from the same subject when the subject was known to be disease-free. Other available sources for the reference level for comparison include aneurysms and adjacent tissue routinely collected during open TAA repair surgery, and blood, plasma, serum, or other body fluid samples collected prior to surgery. Thus, in some embodiments, the level of H4B in a test sample obtained from a subject is compared to the normal standard level of H4B and the known abnormal levels obtained from TAA samples.

[0037] Optionally, the method may further include identifying the subject as a candidate for further testing or monitoring of TAA, e.g., by ultrasound or by repeating the test for H4B after one or more specified time intervals. For example, subjects with more severe cases may be monitored monthly, while those with milder cases may be monitored every three months. The attending physician will be able to adjust this schedule based on the needs and risks of the individual patient. Thus, the method may be repeated, and the measured amount of H4B may be compared to a standard or to previous measurements from the same subject. Initial monitoring may include repeating the test for H4B, and after the H4B test indicates significant progression towards TAA, the subject may be referred for treatment and / or ultrasound evaluation. Measuring the change in the H4B level may detect TAA before it is detected by ultrasound. Early detection of TAA allows for less invasive treatment and avoidance of surgery. For example, if a subject exhibits an initial decrease of 5 - 10% and is later observed to show a 15% decrease after a follow-up period, then the subject needs to be monitored more frequently compared to those subjects with stable levels that do not show any decrease or a small decrease of < 10% over time.

[0038] Also provided is a method for monitoring the therapeutic efficacy of a TAA in a subject. In one embodiment, the method comprises (a) measuring the amount of H4B present in a first test sample obtained from the subject at a first time point; (b) measuring the amount of H4B present in a second test sample obtained from the subject at a second time point; and (c) comparing the measured amounts of H4B present in the first and second test samples. In one embodiment, the method comprises (a) contacting a first test sample obtained from the subject at a first time point with an assay device capable of measuring the amount of H4B present in the test sample; (b) contacting a second test sample obtained from the subject at a second time point with an assay device capable of measuring the amount of H4B present in the test sample; and (c) comparing the measured amounts of H4B present in the first and second test samples. In one embodiment, the method comprises detecting an insufficient increase in H4B present in a second sample compared to a first pre-treatment or early-treatment sample. Preferably, the same or substantially the same assay device and conditions are used to obtain the first and second test samples.

[0039] A treatment is administered to the subject prior to the second time point, and an increase in the amount of H4B present in the second test sample compared to the first test sample indicates an effective treatment of the TAA. This method can be initiated at the start of treatment or after a treatment regimen has been executed. In some embodiments, a statistically significant increase in the amount of H4B present in the second sample compared to the first sample indicates an effective treatment of the TAA. In other embodiments, the increase in H4B is at least about 10% greater than that in the first sample, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80%, 90%, 100%, 150%, 200% or more. The method optionally further comprises prescribing a modified TAA treatment for a subject in whom H4B has decreased or increased compared to the first sample or compared to a standard. For example, the treatment can be modified by increasing or decreasing the amount of folic acid or other therapeutic agent administered to the subject.

[0040] In addition, the present invention provides a method for assessing the severity of a thoracic aortic aneurysm (TAA) or the risk of TAA in a subject. In one embodiment, the method comprises (a) optionally, contacting a test sample from the subject with an assay device capable of measuring the amount of H4B present in the test sample; (b) measuring the amount of H4B present in the test sample; and (c) comparing the measured amount of H4B present in the test sample with the measured amount of H4B present in a standard. The degree of reduction in the amount of H4B present in the test sample as compared to the standard indicates the severity or risk of TAA in the subject. In some embodiments, a statistically significant reduction in the amount of H4B present in the test sample as compared to the standard indicates TAA or susceptibility to TAA. In other embodiments, the reduction in H4B is at least about 10% less than the standard, or at least 20%, 30%, 40%, 50% or 60%, 70%, 80% or 90% less. A smaller reduction generally indicates susceptibility to TAA or a less severe case of TAA, while a larger reduction is more likely to indicate the presence of TAA or a more severe case of TAA. Depending on the amount of H4B present in the test sample, the subject can be monitored or treated as described herein.

[0041] In typical embodiments of the present invention, the sample comprises serum, plasma or whole blood, but the sample can be any body fluid. In a typical example, 2 ml of whole blood is drawn from the subject, although less than about 0.5 ml may be sufficient. The sample can be collected under a variety of conditions, including with or without a preparatory spin down at the time of collection. The sample can be processed using, for example, a variety of collection tubes, including those without supplements, those containing EDTA, those containing heparin and other conditions known in the art. Similarly, the sample can be collected and stored under a variety of conditions, including, for example, by snap freezing in liquid nitrogen and then transferring to -70°C or -80°C; or freezing at -70°C or -80°C and storing the sample there.

[0042] Assay Device and Kit

[0043] In typical embodiments, the assay device comprises a high performance liquid chromatography (HPLC) column, or an immunoassay kit, such as an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescence assay kit or other conventional assay kits. In typical embodiments, the HPLC is equipped with a fluorescence or electrochemical detector and a C-18 column.

[0044] For use in the methods described herein, kits are also within the scope of the present invention. Such kits can include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each containing one of the separate elements to be used in the method. For example, one or more containers can include one or more reagents for detecting H4B optionally labeled in a detectable manner. The kit can also include one or more containers for reporter agent tools, such as biotin-binding proteins, e.g., avidin or streptavidin, which bind to a detectable label, such as an enzyme, fluorescent, or radioisotope label, for detecting H4B.

[0045] The kits of the present invention will generally include the containers described above and one or more other containers that include materials desired from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Additionally, labels can be provided on the containers to indicate that the composition is for a specific application and can also indicate guidelines for use. The guidelines and / or other information can also be included on an insert accompanying the kit.

[0046] Administration and Dosage

[0047] The compositions are often administered in any suitable manner, together with a pharmaceutically acceptable carrier, excipient, or in the form of a pharmaceutically acceptable salt. In the context of the present invention, suitable methods for administering the treatment to a subject are available, and although more than one route can be used to administer a particular composition, a particular route often provides a more direct and effective response compared to another route.

[0048] In the context of the present invention, the dose administered to a patient should be sufficient to achieve a beneficial therapeutic response in the patient over time or sufficient to inhibit disease progression. Accordingly, the composition is administered to the subject in an amount sufficient to elicit an effective response and / or mitigate, alleviate, cure, or at least partially arrest the symptoms and / or complications of the disease. The amount sufficient to achieve this goal is defined as a "therapeutically effective dose". Generally, for a pharmaceutical composition containing folic acid, the amount present in the dose is in the range of about 1 to about 100 milligrams and higher per kilogram of subject body weight. Representative amounts include, but are not limited to, 1, 5, 15, 30, 100 milligrams or higher per kilogram of body weight. The suitable amount will vary with the size of the patient but will generally be in the range of about 1 - 20 milligrams / tablet or 0.1 milliliters to about 5 milliliters.

[0049] The route, frequency, and dosage of administration of the therapeutic compositions disclosed herein will vary from individual to individual and can be readily established using standard techniques. Generally, the pharmaceutical compositions can be administered orally, or by injection (e.g., intradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by inhalation). Typically, at least 1 to 10 doses can be administered over a period of 52 weeks. Preferably, 6 doses are administered at 1-month intervals, and additional supplements can then be given periodically. Alternative regimens may be suitable for individual patients. In one embodiment, 2 or more oral supplements are administered 10 days apart. When treating with folic acid, it is generally best to take it daily, especially for TAA patients. For subjects at risk of TAA who have not yet developed TAA, a lower frequency of administration may be sufficient.

[0050] Generally, appropriate dosages and treatment regimens provide one or more active agents in an amount sufficient to provide a therapeutic and / or prophylactic benefit. Such response can be monitored by establishing improved clinical outcomes in treated patients compared to untreated patients, including by monitoring the values of biomarkers over the course of treatment.

[0051] Treatment includes prophylaxis and therapy. Prophylaxis or therapy can be achieved by a single administration at a single or multiple time points to a single or multiple sites. In some embodiments, the administration is oral. The administration can also be to multiple sites almost simultaneously. The patient or subject includes mammals such as humans, cattle, horses, dogs, cats, pigs, and sheep animals. The subject is preferably human.

[0052] Exemplary Embodiment

[0053] Embodiment 1: A method for detecting thoracic aortic aneurysm (TAA) or susceptibility to TAA in a subject, the method comprising: (a) measuring the amount of tetrahydrobiopterin (H4B) present in a test sample; and (b) comparing the measured amount of H4B present in the test sample with a standard amount of H4B; wherein a decrease in the amount of H4B present in the test sample as compared to the standard indicates TAA or susceptibility to TAA.

[0054] Embodiment 2: The method of Embodiment 1, wherein the measuring comprises contacting the test sample with an assay device.

[0055] Embodiment 3: The method of Embodiment 2, wherein the assay device comprises an immunoassay kit.

[0056] Embodiment 4: The method of Embodiment 2, wherein the assay device comprises a high performance liquid chromatography (HPLC) column.

[0057] Embodiment 5: The method of Embodiment 1, wherein a 20% decrease in the amount of H4B present in the test sample compared to the standard indicates TAA or susceptibility to TAA.

[0058] Embodiment 6: The method of Embodiment 1, wherein a 50% decrease in the amount of H4B present in the test sample compared to the standard indicates TAA or susceptibility to TAA.

[0059] Embodiment 7: The method of Embodiment 1, the method further comprising prescribing TAA treatment to a subject with a decrease in H4B compared to the standard.

[0060] Embodiment 8: The method of Embodiment 7, wherein the treatment comprises folic acid therapy.

[0061] Embodiment 9: The method of Embodiment 8, wherein the folic acid therapy comprises oral administration of folic acid.

[0062] Embodiment 1: The method of Embodiment 1, wherein the sample comprises plasma, serum, or whole blood.

[0063] Embodiment 10: A method for monitoring the therapeutic efficacy of TAA in a subject, the method comprising: (a) contacting a first test sample obtained from the subject at a first time point with a measuring device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; (b) contacting a second test sample obtained from the subject at a second time point with a measuring device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; and (c) comparing the measured amounts of tetrahydrobiopterin (H4B) present in the first test sample and the second test sample; wherein a treatment is administered to the subject before the second time point, and wherein an increase in the amount of H4B present in the second test sample compared to the first test sample indicates effective treatment of TAA.

[0064] Embodiment 11: The method of Embodiment 10, wherein the measuring device comprises a high performance liquid chromatography (HPLC) column.

[0065] Embodiment 12: The method of Embodiment 10, wherein the measuring device comprises an immunoassay kit.

[0066] Embodiment 13: The method of Embodiment 10, wherein a 20% increase in the amount of H4B present in the second test sample compared to the first test sample indicates effective treatment of TAA.

[0067] Embodiment 14: The method of Embodiment 10, wherein the sample comprises plasma, serum, or whole blood.

[0068] Embodiment 15: The method of Embodiment 10, the method further comprising prescribing a modified TAA treatment to a subject in whom H4B is decreased or increased compared to the standard.

[0069] Embodiment 16: A method for assessing the severity or risk of thoracic aortic aneurysm (TAA) in a subject, the method comprising: (a) contacting a test sample from the subject with an assay device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; and (b) measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; (c) comparing the measured amount of H4B present in the test sample with the measured amount of H4B present in a standard; wherein the degree of decrease in the amount of H4B present in the test sample compared to the standard indicates the severity or risk of TAA in the subject.

[0070] Embodiment 17: The method of Embodiment 16, wherein the assay device comprises a high performance liquid chromatography (HPLC) column.

[0071] Embodiment 18: The method of Embodiment 16, wherein the assay device comprises an immunoassay kit.

[0072] Embodiment 19: The method of Embodiment 16, wherein the sample comprises serum or whole blood.

[0073] Embodiment 20: The method of Embodiment 16, the method further comprising prescribing a TAA treatment to a subject in whom H4B is decreased compared to the standard.

[0074] Embodiment 21: The method of Embodiment 16, wherein a 50% decrease in the amount of H4B present in the test sample compared to the standard indicates severe TAA.

[0075] Embodiment 22: A method for treating thoracic aortic aneurysm (TAA) or susceptibility to TAA in a subject, the method comprising: (a) measuring the amount of tetrahydrobiopterin (H4B) present in a test sample of serum, plasma or whole blood from the subject; wherein a 20% decrease in the measured amount of H4B present in the test sample is detected relative to the standard amount of H4B; and (b) if a 20% decrease in the measured amount of H4B present in the test sample is detected relative to the standard amount of H4B, then treating the subject's TAA by oral administration of folic acid therapy.

[0076] Embodiment 23: The method of Embodiment 22, wherein the decrease is a 30% decrease in the amount of H4B present in the test sample compared to the standard.

[0077] Embodiment 24: The method of Embodiment 22, wherein the reduction is a 40% reduction in the amount of H4B present in the test sample compared to the standard.

[0078] Embodiment 25: The method of Embodiment 22, wherein the reduction is a 50% reduction in the amount of H4B present in the test sample compared to the standard.

[0079] Example

[0080] The following examples are presented to illustrate the invention and to assist one of ordinary skill in the art in making and using the invention. The examples are not intended to limit the scope of the invention in any other way.

[0081] Example 1: Tetrahydrobiopterin as a Marker for TAA Formation

[0082] This example demonstrates that reduced levels of tetrahydrobiopterin (H4B) can be used as a biomarker for TAA formation. In a large cohort of TAA patients recruited at the UCLA Ronald Regan Medical Center, a significant decrease in the levels of circulating biomarkers was found in TAA patients ( Figure 1 ). Blood and plasma H4B levels were measured in controls and patients with thoracic aortic aneurysm (TAA). Compared to the control group, blood and plasma H4B levels were significantly lower in TAA patients (from UCLA and NIH NDRI). These results were shown to be statistically significant (p < 0.001) by one-way ANOVA.

[0083] Thus, although the two types of aneurysms, AAA and TAA, have distinct characteristics and well-documented differential mechanistic insights, the data presented here suggest that the underlying mechanism of thoracic aortic aneurysm is downstream of genetic factors such that the same biomarker can be applicable for the detection and treatment of both types of aneurysms.

[0084] Example 2: Prevention of Thoracic Aortic Aneurysm Formation by Dietary Folic Acid

[0085] This example demonstrates that the administration of folic acid (FA) can be used for the treatment and prevention of TAA formation. This study examined whether eNOS uncoupling mediates the formation of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) in Fbn1 C1039G / + Marfan syndrome (MFS) mice, and whether and how countermeasures against TGFβ signaling and eNOS uncoupling can attenuate Marfan aneurysm. Briefly, Fbn1 was treated with FA diet or TGFβ neutralizing antibody (anti-TGFβ) C1039G / +Mice. The diameters of the aortic root and abdominal aorta were measured using echocardiography, while aortic superoxide and nitric oxide (NO) levels were determined by electron spin resonance. The aortic and circulating levels of tetrahydrobiopterin (H4B) were determined using HPLC. Protein expression of NOX4 and inactive / active forms of TGFβ were measured by Western blotting. Results showed that the FA diet significantly attenuated aortic root and abdominal aorta dilation in Fbn1 C1039G / + mice, accompanied by upregulation of the expression and activity of the H4B salvage enzyme dihydrofolate reductase (DHFR), restoration of tissue and circulating levels of H4B, re-coupling of eNOS, and increased bioavailability of NO. Circulating H4B levels accurately predicted tissue H4B bioavailability and were negatively correlated with aortic root dilation. Thus, circulating H4B levels serve as a novel biomarker for the development of TAA and response to treatment. Fbn1 C1039G / + mice had upregulation of mature / active TGFβ and its downstream effector NOX4 expression, but this was attenuated in vivo by anti-TGFβ treatment, allowing eNOS re-coupling and attenuating aneurysm formation. These data for the first time reveal that uncoupled eNOS represents a central mediator of TAA formation in Fbn1 C1039G / + MFS mice, and that the FA diet or TGFβ antagonism abrogates aneurysm formation via re-coupling of eNOS. These data also establish a new Fbn1 / TGFβ / NOX4 / eNOS uncoupling axis in the development of Marfan aneurysms, and targeting this uncoupling axis may contribute to the development of novel therapeutics for treating TAA.

[0086] Aortic aneurysms are associated with significant morbidity and mortality, accounting for 1-2% of all deaths in industrialized countries 1 , causing over 16,450 deaths per year in the United States 2 . Compared to abdominal aortic aneurysms (AAA), aneurysmal disease in humans has a strong genetic influence, particularly for thoracic aortic aneurysms (TAA) 3 . Hereditary thoracic aortic aneurysms and dissections (HTAAD) include Marfan syndrome (MFS), Loeys-Dietz syndrome (LDS), vascular Ehlers-Danlos syndrome (vEDS), and other HTAAD disorders 3-6 . Exploration of the genetic roots of the disease has identified cellular and molecular events such as the uncontrolled release of TGFβ and activation of the TGFβ pathway in MFS. Dysregulation of TGFβ is believed to induce the secretion of matrix-degrading enzymes such as MMP, which represents a common pathway for aneurysm formation 3 .

[0087] Mutations in fibrillin-1 (FBN1) have been established as the cause of aneurysm formation in patients with MFS8 Fibrillin-1 is the main component of 10-nm microfibrils and serves as a scaffold for elastin deposition, providing load-bearing and anchoring functions within the arterial wall. 9 The manifestations of MFS involve multiple organ systems, including the aorta, heart and valves, bones, eyes, lungs, and dura mater. 10 In 1955, Dr. McKusick first described the cardiovascular features of MFS. 11 The main cardiovascular abnormality is an aneurysm of the aortic root, which often extends to the proximal part of the tubular ascending thoracic aorta to form a pear-shaped aortic dilation. 3 Aortic aneurysms and dissections are the most life-threatening manifestations of MFS. 4,12 .

[0088] In different animal models, the role of TGFβ in TAA is different or controversial. 5,12,13 MFS mice with non-anatomical TAA (Fbn1 C1039G / + mice) develop aneurysms due to overstimulation of TGFβ production and signaling caused by inappropriate activity of AT1R. 5,14 In addition, in Fbn1 C1039G / + mice, atypical (smad-independent) TGFβ signaling was found to be the main driver of aortic disease. 15 However, MFS mice with a more severe phenotype (Fbn1 mgR / mgR mice) showed a harmful effect of TGF-β inhibition on TAA. It has been reported that anti-TGFβ neutralizing antibodies enhanced aortic rupture and aneurysms in the chest and abdomen of Ang II-infused C57BL / 6J mice. 16 .

[0089] A key role of oxidative stress has been demonstrated in the pathogenesis of AAA. 17-22 Regarding TAA, evidence indicates a correlation between oxidative stress and TAA in humans. 23 In human TAA tissues, the in-situ production of ROS and the expression of the NADPH oxidase subunit p22phox were significantly increased, and statins and AT1R blockers (ARBs) could inhibit aneurysm formation by inhibiting p22phox. 23 The expression of NOX4 was increased in the media of human Marfan aorta and transcriptionally upregulated in VSMCs. 24 Notably, Fbn1 C1039G / + -NOX4 - / - double mutant mice showed a reduction in fragmented elastic fibers in the middle segment of the aorta, accompanied by an improvement in Marfan-related enlargement of the aortic root. 24In addition, NOX4 deletion in Marfan mice exacerbated the thickening of the middle cerebral artery (MCA) wall, accompanied by increased deposition of collagen 25 。

[0090] Recent work has established a direct causal role for uncoupled eNOS and endothelium-derived reactive oxygen species (ROS) in AAA formation in both novel and classical AAA models, including Ang II-infused hph-1 mice and Ang II-infused apoE knockout mice 19-22 。Moreover, oral administration of folic acid (FA) to re-couple eNOS completely or largely attenuated AAA formation in these animals 21,22 。Accordingly, the following hypothesis was tested: that eNOS uncoupling is induced by an initial TGFβ-dependent increase in ROS production, leading to aneurysm formation in Fbn1 C1039G / + mice, and that targeting uncoupled eNOS with FA diet or targeting TGFβ signaling with anti-TGFβ antibody effectively prevents Marfan aneurysm by attenuating NOX4 expression. Fbn1 C1039G / + mice were treated with FA diet or TGFβ-neutralizing antibody (anti-TGFβ). In Fbn1 C1039G / + mice, the diameters of the aortic root and abdominal aorta increased in an age-dependent manner, and this increase was greatly attenuated by administration of FA. This was associated with a marked increase in tissue and circulating H4B levels, re-coupling of eNOS, and improved NO bioavailability. Circulating H4B levels were closely correlated with tissue levels and the size of the aortic root, indicating a novel biomarker role for circulating H4B in TAA development and response to treatment. In Fbn1 + / + and Fbn1 C1039G / + mice, the expression and activity of endothelial DHFR were substantially upregulated, thus restoring H4B levels. Notably, the baseline deficiency of H4B levels in Fbn1 C1039G / + mice was due to decreased expression of GTPCHI in the mice. Compared with Fbn1 + / + mice, the expression of mature TGFβ and its downstream effector NOX4 was elevated in Fbn1 C1039G / + mice. In vivo treatment with anti-TGFβ eliminated NOX4 expression, re-coupled eNOS, and attenuated aortic root dilation. Thus, the TGFβ / NOX4 / eNOS uncoupling axis represents a new molecular pathway for TAA formation in Fbn1 C1039G / + mice, and targeting this pathway may contribute to the development of novel therapeutic agents for Marfan aneurysm and other types of TAA

[0091] Materials and Methods

[0092] Chemical reagents

[0093] Unless otherwise noted, all chemicals and reagents were purchased from Sigma-Aldrich at the highest purity. Isoflurane was purchased from Piramal Healthcare.

[0094] Animals

[0095] All experimental procedures were approved by the Institutional Animal Care and Usage Committee of the University of California, Los Angeles (UCLA). The original heterozygous Fbn1 C1039G / + male animals were purchased from Jackson Labs (Bar Harbor, ME, strain B6.129-Fbn1 tm1Hcd / J, stock number 012885). Heterozygous mice developed proximal aortic aneurysms, mitral valve thickening, alveolar septation defects, mild thoracic kyphosis, and myopathy, but 90% were reported to survive to 1 year of age. This strain was backcrossed to C57BL / 6 for more than 9 generations by the donor laboratory 14 . All pups were genotyped by PCR according to the instructions of Jackson Labs ( Figure 2 ).

[0096] Folic acid treatment

[0097] For the group of animals treated with folic acid (FA), the standard diet was replaced with an in-house custom-made food tablet containing FA (15 mg / kg / day), which has been shown to restore eNOS re-coupling by restoring the function of dihydrofolate reductase (DHFR) to increase the bioavailability of tetrahydrobiopterin 21,22,26 . FA treatment began at 4 weeks of age and continued throughout the 8-week study period until collection.

[0098] Ultrasonographic imaging of the aortic root and abdominal aorta

[0099] Animals were anesthetized with isoflurane (approximately 1.5%) and secured to a temperature-controlled table to maintain the temperature at 37°C. Hair was removed from the abdomen and chest with depilatory cream (Nair). Pre-warmed ultrasonic transmission gel was applied to the chest (for the aortic root) or abdomen (for the abdominal aorta). The ultrasonic probe (Velvo 2100, echocardiograph, MS-400) was placed on the gel to visualize the aorta transversely. For the abdominal aorta, the aorta was first confirmed by identifying pulsatile flow using Doppler measurement. Consistent positioning for image acquisition was ensured by imaging the area immediately above the left renal artery branch. For the aortic root, the aorta immediately above the heart was imaged, and Doppler measurement was used to confirm the aorta. All images were recorded and saved for later offline aortic size analysis.

[0100] Measurement of superoxide using electron spin resonance (ESR)

[0101] As previously described, aortic superoxide was measured by ESR 19 -22 ,26-33 Briefly, freshly isolated aorta was homogenized in ice-cold lysis buffer containing a 1:100 protease inhibitor mixture and centrifuged at 12,000 g for 15 minutes. Protein content of the supernatant was determined using a protein assay kit (Bio-Rad, #500-0113, #500-0114, #500-0115). 5 μg of protein was mixed with ice-cold and nitrogen-bubbled Krebs / HEPES buffer containing diethyldithiocarbamic acid (5 μmol / L), deferoxamine (25 μmol / L), and the superoxide-specific spin trap methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, 500 μmol / L, Axxora, San Diego, CA, USA). The mixture was then loaded into a glass capillary (Kimble, Dover, OH, USA) and peroxide production was measured using an ESR spectrometer (eScan, Bruker, Billerica, MA, USA). PEG-SOD (100 U / mL) was added for a second measurement to determine background. To assess eNOS uncoupling, L-NAME (100 μmol / L) was added for a third measurement. The ESR settings used were: center field, 3480; sweep width, 9 G; microwave frequency, 9.78 GHz; microwave power, 21.02 mW; modulation amplitude, 2.47 G; 512-point resolution; receiver gain, 1000.

[0102] Measurement of nitric oxide (NO) using electron spin resonance

[0103] As previously described, aortic NO production was also measured using ESR 19 -22 ,26 -29 ,32-34 Briefly, freshly isolated aorta was cut into 2-mm rings and then, in the presence of the calcium ionophore A23187 (10 μmol / L), at 37 °C in a solution containing freshly prepared NO-specific spin trap Fe 2+Incubate in nitrogen-bubbled modified Krebs / HEPES buffer (as described above) with (DETC)2 (0.5 mmol / L) for 60 minutes. Then freeze the aortic rings rapidly in liquid nitrogen and place them in a finger Dewar for ESR measurement. The instrument settings are as follows: center field, 3440; scan width, 100 G; microwave frequency, 9.796 GHz; microwave power, 13.26 mW; modulation amplitude, 9.82 G; 512-point resolution; and receiver gain, 356.

[0104] Measure tetrahydrobiopterin (H4B) using HPLC

[0105] Measure aortic H4B and plasma H4B using HPLC as previously described 19 -22 ,26,27,32,33,35-37 . For the aorta, lyse freshly isolated aorta in H4B lysis buffer (0.1 M phosphoric acid, 1 mM EDTA, 10 mM dl-dithiothreitol), then centrifuge at 12,000 g for 3 minutes at 4 °C in the dark. For plasma, mix an equal volume of plasma and H4B lysis buffer and incubate on ice for 20 minutes in the dark, then centrifuge at 12,000 g for 3 minutes at 4 °C in the dark. Oxidize the supernatants of the aorta and plasma in acidic solution (0.2 M trichloroacetic acid containing 2.5% I2 and 10% KI) and basic solution (0.1 M NaOH containing 0.9% I2 and 1.5% KI). After centrifugation, inject 10 μl of the supernatant into an HPLC system equipped with a fluorescence detector (SHIMADZU AMERICA INC, Carlsbad, CA, USA). Detect H4B and its oxidized species using excitation and emission wavelengths of 350 nm and 450 nm. Calculate the H4B concentration as previously described 38,39 .

[0106] Western blotting

[0107] Protein blotting was performed using a 12.5% SDS / PAGE gel and nitrocellulose membrane following standard protocols. The primary antibodies used and their dilutions were: DHFR (1:500, Novus Biologicals, H00001719-M01), β-actin (1:3000, Sigma-Aldrich, A2066), eNOS (1:2000, BD Transduction Laboratories, 610297), TGFβ (1:500, Abcam, ab92486), NOX4 (1:300, Novus Biologicals, NB110-58849SS), and GTPCH I (1:500, Abbiotec, 250680).

[0108] DHFR activity was determined using HPLC

[0109] DHFR activity was measured from isolated EC or lysates of denuded aortic rings as previously described 19,22,26 . Briefly, lysates were incubated with NADPH (200 μmol / / L) and the substrate dihydrofolate (50 μmol / L) in DHFR assay buffer (0.1 mol / L dipotassium hydrogen phosphate, 1 mmol / L DTT, 0.5 mmol / L KCl, 1 mmol / L EDTA, and 20 mmol / L sodium ascorbate, pH 7.4) for 20 minutes at 37 °C in the dark. An HPLC system (SHIMADZU AMERICA INC, Carlsbad, CA, USA) with a C-18 column (Alltech, Deerfield, MA, USA) was used to measure the reaction product tetrahydrofolate (THF) using an aqueous mobile phase consisting of 7% acetonitrile and 5 mmol / L dipotassium hydrogen phosphate (pH 2.3). Signals were detected using a fluorescence detector at an excitation of 295 nm and an emission of 365 nm. THF content was calculated relative to a standard curve prepared using a THF solution in the assay buffer. Data were presented as nmol THF produced per minute per milligram of protein.

[0110] In vivo treatment with anti-TGFβ antibody

[0111] As previously shown, four-week-old heterozygous Fbn1 male animals were treated with a TGFβ-neutralizing antibody (anti-TGFβ, clone 1D11, Bio X Cell) or isotype (IgG, clone MOPC21, Bio X Cell) C1039G / + male animals 40. On the first day, 1 mg of anti-TGFβ or isotype reagent was injected intraperitoneally, and then 200 μg was injected intraperitoneally every other day for a total of 13 times. As described above, ultrasonic imaging of the aortic root and abdominal aorta was performed weekly. As described above, aortic superoxide production and eNOS uncoupling activity were determined 4 weeks after injection.

[0112] Statistical analysis

[0113] All analyses were performed using Graphpad Prism software. Student’s t-test was used for comparison between two groups. ANOVA, followed by Newman-Keuls post-hoc test, was used for comparison between multiple groups. Statistical significance was set at p < 0.05. All grouped data were presented as mean ± SEM.

[0114] Results

[0115] Oral administration of folic acid re-couples eNOS to inhibit Fbn1 C1039G / + Dilation of the aortic root and abdominal aorta in mice

[0116] Ultrasound images were obtained to examine Fbn1 with or without FA treatment + / + or Fbn1 C1039G / + The size of the aortic root and abdominal aorta in mice. Compared with Fbn1 + / + , Fbn1 C1039G / + mice showed significant dilation of the aortic root and abdominal aorta ( Figure 3A and 3B ). However, the aortic root and abdominal aorta of Fbn1 C1039G / + mice treated with FA diet were much smaller than those of Fbn1 C1039G / + mice fed with a chow diet. These results indicate that FA treatment may be an effective treatment for attenuating aneurysm formation in MFS.

[0117] In Fbn1 C1039G / + mice, folic acid reduces superoxide production, increases NO bioavailability, and re-couples eNOS.

[0118] Consistent with the above findings that FA diet eliminated aneurysm formation in Fbn1 C1039G / + mice and had a significant effect on attenuating AAA formation in various novel and classical mouse models 19-22 , in Fbn1 C1039G / + mice, FA diet was able to reduce superoxide production, increase NO bioavailability, and eliminate eNOS uncoupling activity (Figure 4).

[0119] First, ESR was used to measure aortic superoxide production in the presence or absence of the NOS inhibitor L-NAME. If eNOS is functional and coupled, then inhibition of eNOS by L-NAME will increase the measured superoxide due to the lack of NO scavenging of superoxide. However, if eNOS is dysfunctional and uncoupled, then superoxide is produced and inhibition with L-NAME will reduce the measured superoxide. Thus, the difference in superoxide measured in the presence and absence of L-NAME reflects the coupled / uncoupled state of eNOS. As Figure 4A is evident, in 8-week-old and 12-week-old Fbn1 C1039G / + mice, L-NAME-sensitive superoxide production reflecting eNOS uncoupling activity was significantly increased, and this increase was completely attenuated by oral treatment with FA.

[0120] Since FA restored eNOS function, NO levels were then measured in aortas isolated from Fbn1 + / + and Fbn1 C1039G / + mice with or without oral FA treatment. Figure 4B The results shown in + / + demonstrate that compared to 8-week-old and 12-week-old Fbn1 C1039G / + mice, the bioavailability of NO in the aortas of Fbn1 C1039G / + mice was significantly reduced, while FA significantly increased the NO bioavailability in Fbn1

[0121] These results indicate that FA prevents aneurysm formation by recoupling eNOS to attenuate superoxide production from eNOS and increase NO bioavailability.

[0122] Folic acid restored tissue and circulating H4B levels in Fbn1 C1039G / + animals.

[0123] Uncoupling of eNOS is caused by reduced bioavailability of H4B, a cofactor required for proper eNOS coupling activity 21,26,28,29,41-43 . Thus, to further examine the coupled state of eNOS, the bioavailability of H4B was determined by HPLC in aortic and plasma samples from 4-, 8-, and 12-week-old Fbn1 + / + and Fbn1 C1039G / + animals. Figure 5A and 5B The results in + / + indicate that compared to 4-week-old Fbn1 C1039G / +The levels of H4B in the aorta and plasma of mice were significantly reduced. After oral treatment with FA, compared with mice fed a chow diet, Fbn1 + / + and Fbn1 C1039G / + In the aorta of mice ( Figure 5A ) and plasma ( Figure 5B ), the levels of H4B were substantially restored, indicating that the coupling state of eNOS was improved under FA treatment.

[0124] Recent studies have shown that circulating H4B can be used as a novel biomarker for AAA 35 . Here, circulating H4B levels were also detected in the plasma of Fbn1 C1039G / + mice with or without FA treatment. As Figure 5B shown, the changes in H4B levels in plasma were consistent with the changes in the aorta ( Figure 5A ). The linear correlation between tissue and plasma H4B was calculated for the above data. Figure 5C It was shown that the circulating H4B levels were closely correlated with the tissue H4B levels. In Fbn1 C1039G / + animals, the reduced H4B levels were closely correlated with the larger aortic root diameter ( Figure 5D and 5E ).

[0125] These results indicate that the deficiency of H4B is involved in eNOS uncoupling-dependent aneurysm development, and this development was reversed by the FA diet. Additionally, circulating H4B levels can be used clinically as a strong biomarker for the development and treatment response of TAA.

[0126] Oral folic acid treatment preserved the DHFR expression and activity in the aorta of Fbn1 C1039G / + mice.

[0127] The above data show that the restoration of eNOS coupling related to the bioavailability of H4B may play an important role in the protective effect of FA against TAA in MFS animals. Previous studies have shown that FA treatment can recouple eNOS by improving the endothelial DHFR expression and activity in AAA, which is essential for rescuing H4B 21,22 . Here, the endothelial DHFR activity and expression in Fbn1 C1039G / + mice were examined to test whether DHFR was also improved during FA prevention of TAA in this model.

[0128] Endothelial cells (ECs) were isolated from freshly prepared aortas, protein blotting was used to detect DHFR expression in aortic ECs, and HPLC was performed to assess Fbn1 + / + and Fbn1 C1039G / +DHFR activity in isolated ECs and denuded aortas of animals. Figure 6A Representative western blots of eNOS (144 kD), β-actin control (42 kD), and DHFR (21 kD) are shown. After FA treatment, Fbn1 + / + and Fbn1 C1039G / + expression of DHFR ( Figure 6B ) rather than eNOS ( Figure 6C ) increased in animals. As Figure 6D and 6E shown, DHFR activity was measured by HPLC in isolated ECs ( Figure 6D ) and denuded aortas ( Figure 6E ) of animals. By oral FA treatment, DHFR activity in isolated ECs and denuded aortas of Fbn1 C1039G / + and Fbn1 + / + mice increased significantly.

[0129] These results clearly indicate that in Fbn1 C1039G / + mice, FA restores eNOS activity to attenuate TAA, accompanied by a marked upregulation of DHFR expression and activity.

[0130] Notably, although the H4B level was decreased in Fbn1 + / + mice compared with Fbn1 C1039G / + mice ( Figure 5A and 5B ), DHFR expression ( Figure 6A and 6B ) was unchanged at baseline. The expression levels of the rate-limiting H4B synthase GTPCHI were examined in these animals ( Figure 7A and 7B ), and a marked decrease in GTPCHI expression was found in Fbn1 C1039G / + mice at baseline. Thus, this regulation seems to underlie the decreased H4B level at baseline, while the beneficial effect of FA on increasing the bioavailability of H4B to re-couple eNOS is mediated by a substantial restoration of DHFR expression and activity.

[0131] Anti-TGFβ attenuates aortic root dilation by downregulation of NOX4 and re-coupling of eNOS in Fbn1 C1039G / + animals

[0132] TGFβ signaling has been found to play a crucial role in the development and maintenance of the vascular system; mutations in genes related to the TGFβ signaling pathway lead to MFS 4 . Treatment with anti-TGFβ has been reported to prevent aortic aneurysm in a mouse MFS model by modulating the canonical TGFβ signaling pathway 14。It was also found that atypical TGFβ signaling is involved in the progression of aortic aneurysms in MFS mice 15 。In addition, during aneurysm formation and progression in Fbn1 MFS mice, TGFβ was found to strongly induce NOX4 24 。In primary cultures of rat VSMCs in vitro, TGFβ also increased NOX4 expression and ROS production 44 。In AAA 19 、cardiac ischemia / reperfusion (I / R) injury 45 and aging 46 conditions, NOX4 induced eNOS uncoupling in ECs. It was hypothesized that the Fbn1 / TGFβ / NOX4 axis is upstream of uncoupled eNOS in the induction of TAA formation in MFS mice

[0133] Results showed that compared with Fbn1 + / + mice, the protein level of mature TGFβ rather than the protein level of inactive TGFβ was significantly increased in the aorta of Fbn1 C1039G / + mice ([[]] Figure 7C and 7D ). Compared with Fbn1 + / + mice, NOX4, which is downstream of mature TGFβ and known to uncouple eNOS, was also upregulated in Fbn1 C1039G / + mice ([[]] Figure 7E and 7F ).

[0134] In addition, compared with the injection of IgG in Fbn1 C1039G / + mice, anti-TGFβ antibody reduced NOX4 expression ([[]] Figure 8A and 8B ). After injecting anti-TGFβ antibody into Fbn1 C1039G / + mice for 3 weeks and 4 weeks, the aortic root diameter decreased ([[]] Figure 8C ), while there was still a significant difference in the abdominal aortic diameter between the anti-TGFβ injection group and the IgG injection group ([[]] Figure 8D ). The latter may be related to the relatively slow progression of AAA in Fbn1 C1039G / + mice. The eNOS uncoupling activity was determined after injecting anti-TGFβ antibody into Fbn1 C1039G / + mice. As shown in Figure 8E , in vivo anti-TGFβ antibody treatment completely re-coupled eNOS in Fbn1 C1039G / + .

[0135] In summary, these data established that mediate Fbn1 C1039G / +A new TGFβ-NOX4-eNOS uncoupling axis is formed by TAA in mice, and eNOS re-coupling by FA diet or anti-TGFβ treatment can prevent TAA by targeting the components of this pathway.

[0136] Discussion

[0137] The most important finding of this study is the first demonstration of the causal role of eNOS uncoupling, and the therapeutic potential of re-coupling eNOS by targeting the Fbn1 / TGFβ / NOX4 axis in the formation of TAA in MFS mice. In Fbn1 C1039G / + mice, FA significantly reduced the diameters of the aortic root and abdominal aorta through the DHFR / H4B / eNOS re-coupling / NO pathway ( C1039G / + ). Circulating H4B accurately reflects aortic H4B levels, and aortic and circulating H4B levels are negatively correlated with the diameters of the aortic root and abdominal aorta. In Fbn1 Figure 9 mice, the expression of mature TGFβ and its downstream effector NOX4 is increased, while in vivo treatment with anti-TGFβ antibody reduces NOX4 expression, re-couples eNOS and reduces the diameter of the aortic root ( C1039G / + ). Therefore, oral FA treatment and anti-TGFβ targeting eNOS re-coupling may represent new strategies for treating TAA in Fbn1 Figure 9 mice and other types of TAA. C1039G / +

[0138] It has been confirmed that oxidative stress plays a crucial role in the pathogenesis of aortic aneurysms, especially AAA 17-22,47 . Previous work has elucidated that FA completely or largely attenuates AAA formation through the re-coupling of eNOS 21,22 . This example examined whether oxidative stress and uncoupled eNOS are the causes of TAA formation in Fbn1 C1039G / + mice, and comprehensively explored whether FA, a potential oral drug for the treatment of TAA in Fbn1 C1039G / + mice, can be used. Previous studies have revealed a positive correlation between oxidative stress and the severity of TAA 23,48-50 . The overproduction of ROS has been considered a pathogenic mechanism for aortic aneurysms and other manifestations occurring in MFS 23,51,52 . Here, it was found that eNOS uncoupling occurs in TAA of Fbn1 C1039G / + mice to produce superoxide ( Figure 4A ), and this is the first evidence that eNOS uncoupling serves as the main source of ROS for forming TAA ( Figure 3A ). Surprisingly, these results are in contrast to AAA Figure 3A 19-22,32and cardiac ischemia / reperfusion (I / R) injury 45,53,54 similar to those found in. Since oral administration of FA can restore the coupled activity of eNOS to prevent AAA formation in novel and classical AAA models including Ang II-infused hph-1 mice and Ang II-infused apoE knockout mice 21,22,26 , the ability of FA to similarly robustly re-couple eNOS to attenuate TAA formation was investigated in Fbn1 C1039G / + mice. Results showed that FA fully restored the coupled activity of eNOS to increase NO bioavailability, thereby abolishing dilation of the aortic root and abdominal aorta in Fbn1 C1039G / + mice. These data demonstrate that FA diet may represent a novel therapeutic strategy for TAA by restoring the coupled activity of eNOS.

[0139] Deficiency of H4B converts eNOS from a coupled state to a de-coupled state 21,26,28,29,41-43 . This study found that aortic and circulating H4B levels were greatly reduced in Fbn1 C1039G / + ( Figure 5A and 5B ), accompanied by de-coupled activity of eNOS. Results further showed that oral administration of FA restored the bioavailability of H4B in tissues and plasma( Figure 5A and 5B ), which was associated with abolishing de-coupled activity of eNOS and preventing TAA formation in Fbn1 C1039G / + mice. Notably, aortic and plasma H4B levels were quantitatively correlated with the size of the aortic root, with lower H4B levels corresponding to larger aortic root dimensions. Thus, the current data provide the first evidence for the biomarker role of circulating H4B in TAA formation in Fbn1 C1039G / + mice.

[0140] Over the past decade, substantial work has established the important role of the H4B salvage enzyme DHFR in regulating H4B bioavailability, eNOS coupled / de-coupled activity, and vascular pathogenesis when deficient 19,22,26,28,29,36,55,56 . Deficiency of DHFR induces reduced H4B bioavailability and subsequent eNOS de-coupling, leading to the development of cardiovascular diseases including hypertension, aortic aneurysm, diabetic vascular complications, I / R injury, and heart failure 19-22,26,30,32,33,36,57 . After FA treatment in Fbn1 C1039G / + mice, both the expression and activity of DHFR in aortic ECs were improved, indicating a new observation of DHFR-dependent TAA attenuation. Similar results have been reported in AAA 19-22。Therefore, FA treatment can re-couple eNOS by improving endothelial DHFR function in AAA and TAA. Notably, the expression and activity of DHFR in ECs were not reduced at baseline, while the reduced expression of GTPCHI protein seems to be the cause of basal H4B deficiency in animals. Whether this loss of GTPCHI is directly downstream of Fbn1 deficiency requires further investigation. C1039G / + The mutation of fibrillin-1 leads to the uncontrolled release of TGFβ and the activation of the TGFβ pathway.

[0141] In the aneurysm formation and progression of murine MFS models, TGFβ strongly induces the expression level of NOX4. 7,58,59 Moreover, after NOX4 knockout, the diameter of the aortic root in Fbn1 mice was eliminated. 24 C1039G / + However, the mediating role of NOX4 in aneurysm formation in MFS has not been fully elucidated. This example presents the following new findings: compared with WT littermates, the mature form rather than the inactive form of TGFβ is elevated in Fbn1 mice ( 24 and C1039G ). The expression of NOX4 protein also increases ( Figure 7C and 7D ), and eNOS is de-coupled ( Figure 7C ). Treatment with anti-TGFβ antibody reduces total superoxide production and re-couples eNOS by reducing NOX4 expression ( Figure 4A ), thereby attenuating aortic root dilation in Fbn1 mice ( Figure 8A ). These results indicate that NOX4 is downstream of TGFβ signaling in mediating TAA formation in Fbn1 mice. Similarly, upregulation of NOX4 expression was found in human AAA segments and in the aortas of Ang II-treated ApoE Figure 8E mice. In ApoE Figure 8C mice, AAA formation exacerbated by homocysteine (Hcy) also significantly upregulates NOX4, and NOX4 siRNA reduces Hcy-induced adventitial fibroblast activation. C1039G C1039G / + Knockout of NOX4 reduces the incidence of AAA in Ang II-treated hph-1 mice by re-coupling eNOS. - / - In mice with cardiac ischemia / reperfusion (I / R) injury, NOX4 RNAi reduces the infarct area by re-coupling eNOS. 60 - / - 61 19 45 ​​​​​​。It was also found that NOX4 mediates eNOS uncoupling in vitro. In ECs, NOX4 is mainly responsible for eNOS uncoupling during aging. 46 。In glomerular mesangial cells, inhibition of NOX4 abolished eNOS uncoupling triggered by high glucose or AngII, thus demonstrating that NOX4 activation leads to eNOS uncoupling. 62，63 。Collectively, these findings established that NOX4 / eNOS uncoupling is downstream of TGFβ to mediate TAA formation in Fbn1 C1039G / + mice.

[0142] In summary, the data elucidated that the TGFβ / NOX4 / eNOS uncoupling axis is innovatively the cause of TAA formation in MFS mice, and targeting the uncoupling axis with a FA diet (through the DHFR / H4B / eNOS recoupling / NO pathway) or in vivo anti-TGFβ antibody treatment (by inhibiting NOX4) eliminates TAA formation through eNOS recoupling. These findings provide novel targeted therapeutic agents for the treatment or prevention of human TAA.

[0143] References

[0144] 1. Lindsay ME, Dietz HC. Nature 2011;473:308-316.

[0145] 2. Benjamin EJ, et al. Circulation 2019;139:e56-e66.

[0146] 3. Isselbacher EM, et al. Circulation 2016;133:2516-2528.

[0147] 4. Gillis E, et al. Circulation research 2013;113:327-340.

[0148] 5. Davis FM, et al. Arteriosclerosis.thrombosis, and vascular biology 2019;39:e83-e90.

[0149] 6. Guo J, et al. Scientific reports 2015;5:13115.

[0150] 7. Isogai Z, et al. The Journal of biological chemistry 2003; 278: 2750 - 2757.

[0151] 8. Dietz HC, et al. Nature 1991; 352: 337 - 339.

[0152] 9. Sherratt MJ, et al. Micron 2001; 32: 185 - 200.

[0153] 10. Braverman AC, et al. Circulation 2015; 132: e303 - 309.

[0154] 11. McKV. Circulation 1955; 11: 321 - 342.

[0155] 12. Daugherty A, et al. Journal of the American Heart Association 2017; 6.

[0156] 13. Lu H, Daugherty A. Arteriosclerosis, thrombosis, and vascular biology 2017; 37: e59 - e65.

[0157] 14. Habashi JP, et al. Science 2006; 312: 117 - 121.

[0158] 15. Holm TM, et al. Science 2011; 332: 358 - 361.

[0159] 16. Chen X, et al, PloS one 2016; 11: e0153811.

[0160] 17. Thomas M, et al. Circulation 2006; 114: 404 - 413.

[0161] 18. Gavrila D, et al. Arteriosclerosis, thrombosis, and vascular biology 2005; 25: 1671 - 1677.

[0162] 19. Siu KL, et al., Redox biology 2017; 11: 118 - 125.

[0163] 20. Miao XN, Siu KL, Cai H. Journal of molecular and cellular cardiology 2015; 87: 152 - 159.

[0164] 21. Gao L, et al. Hypertension 2012; 59: 158 - 166.

[0165] 22. Siu KL, et al. PloS one 2014; 9: e88899.

[0166] 23. Ejiri J, et al. Cardiovascular research 2003; 59: 988 - 996.

[0167] 24. Jimenez - Altayo F, et al. Free radical biology & medicine 2018; 118: 44 - 58.

[0168] 25. Onetti Y, et al. American journal of physiology Heart and circulatory physiology 2016; 310: H1081 - 1090.

[0169] 26. Gao L, et al. Journal of molecular and cellular cardiology 2009; 47: 752 - 760.

[0170] 27. Youn JY, et al. Molecular endocrinology 2015; 29: 1123 - 1133.

[0171] 28. Chalupsky K, et al. Proceedings of the National Academy of Sciences of the United States of America 2005; 102: 9056 - 9061.

[0172] 29. Oak JH, et al Diabetes 2007; 56: 118 - 126.

[0173] 30. Youn JY, et al. Diabetologia 2012; 55: 2069 - 2079.

[0174] 31. Youn JY, et al. Diabetes 2014; 63: 2344 - 2355.

[0175] 32. Li Q, et al. Redox biology 2019; 24: 101185.

[0176] 33. Li H, et al. Hypertension 2019; 73: 179 - 189.

[0177] 34. Youn JY, et al. Circulation research 2009; 104: 50 - 59.

[0178] 35. Siu KL, Cai H. American journal of physiology Heart and circulatory physiology 2014; 307: H1559 - 1564.

[0179] 36. Crabtree MJ, et al. The Journal of biological chemistry 2009; 284: 28128 - 28136.

[0180] 37. Chuaiphichai S, et al. British journal of pharmacology 2017; 174: 657 - 671.

[0181] 38. Alp NJ, et al. The Journal of clinical investigation 2003; 112: 725 - 735.

[0182] 39. Cai S, et al. Cardiovascular research 2002; 55: 838 - 849.

[0183] 40. Manlove LS, et al. Journal of immunology 2015; 195: 4028 - 4037.

[0184] 41. Vasquez-Vivar J, et al. Proceedings of the National Academy of Sciences of the United States of America 1998; 95: 9220 - 9225.

[0185] 42. Wever RM, et al. Biochemical and biophysical research communications 1997; 237: 340 - 344.

[0186] 43. Xia Y, et al. The Journal of biological chemistry 1998; 273: 25804 - 25808.

[0187] 44. Liu XH, et al. Free radical biology & medicine 2016; 94: 174 - 184.

[0188] 45. Siu KL, et al. Journal of molecular and cellular cardiology 2015; 78: 174 - 185.

[0189] 46. Lee HY, et al. Free radical biology & medicine 2017; 113: 26 - 35.

[0190] 47. Miller FJ, Jr., et al. Arteriosclerosis, thrombosis, and vascular biology 2002; 22: 560 - 565.

[0191] 48. Branchetti E, et al. Cardiovascular research 2013; 100: 316 - 324.

[0192] 49. Liao M, et al. The Journal of thoracic and cardiovascular surgery 2008; 136: 65 - 72, 72 e61 - 63.

[0193] 50. Fiorillo C, et al. International journal of cardiology 2010; 145: 544 - 546.

[0194] 51. Phillippi JA, et al. Circulation 2009; 119: 2498 - 2506.

[0195] 52. Tabakoglu E, et al. Mediators of inflammation 2004; 13: 209 - 210.

[0196] 53. Hallstrom S, et al. Cardiovascular research 2008; 77: 506 - 514.

[0197] 54. Mendez - Carmona N, et al. The Journal of heart and lung transplantation: the official publication of the International Society for Heart Transplantation 2019.

[0198] 55. Thony B, et al. The Biochemical journal 2000; 347 Pt 1: 1 - 16.

[0199] 56. Michel T. NO way to relax: the complexities of coupling nitric oxide synthase pathways in the heart. Circulation 2010; 121: 484 - 486.

[0200] 57. Cario H, et al. American joumal of human genetics 2011; 88: 226 - 231.

[0201] 58. Dallas SL, et al. The Joumal of cell biology 1995; 131: 539 - 549.

[0202] 59. Dallas SL, et al. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2000; 15: 68 - 81.

[0203] 60. Lu WW, et al. Arteriosclerosis, thrombosis, and vascular biology 2016; 36: 2176 - 2190.

[0204] 61. Liu Z, et al. Circulation research 2012; 111: 1261 - 1273.

[0205] 62. Eid AA, et al. Molecular and cellular biology 2013; 33: 3439 - 3460.

[0206] 63. Lee DY, et al. The Journal of biological chemistry 2013; 288: 28668 - 28686.

[0207] Throughout this application, various publications are referenced. The entire disclosures of these publications are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0208] Those skilled in the art will appreciate that the concepts and specific embodiments disclosed in the foregoing description can be readily used as a basis for modifying or designing other embodiments for achieving the same purposes of this invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of this invention as set forth in the appended claims.

Claims

1. Use of a determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample for manufacturing a determination device for detecting thoracic aortic aneurysm (TAA) in a subject, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample is used to perform step (a): contacting a test sample from the subject with the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; wherein detecting thoracic aortic aneurysm (TAA) in the subject further includes step (b): comparing the measured amount of H4B present in the test sample with a standard amount of H4B; wherein the standard amount of H4B is the amount of H4B in a healthy subject without TAA; wherein a reduction of at least 10% in the amount of H4B present in the test sample as compared to the standard amount of H4B indicates TAA.

2. The use according to claim 1, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample comprises an immunoassay kit or a chemiluminescence assay kit.

3. The use according to claim 1, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample comprises a high performance liquid chromatography (HPLC) column.

4. The use according to claim 1, wherein a reduction of at least 20% in the amount of H4B present in the test sample as compared to the standard amount of H4B indicates TAA.

5. The use according to claim 1, wherein a reduction of at least 40%, 50%, 60% or 80% in the amount of H4B present in the test sample as compared to the standard amount of H4B indicates TAA.

6. The use according to claim 1, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample is further used to prescribe TAA treatment for a subject with a reduction in H4B as compared to the standard amount of H4B.

7. The use according to claim 6, wherein the treatment comprises folic acid therapy or dihydrofolate reductase (DHFR) gene therapy.

8. The use according to claim 7, wherein the folic acid therapy comprises oral administration of folic acid.

9. The use according to claim 1, wherein the sample comprises plasma, serum or whole blood.

10. Use of a determination device capable of measuring the amount of tetrahydrobiopterin H4B present in a test sample for manufacturing a determination device for monitoring the therapeutic efficacy of TAA in a subject, wherein the determination device capable of measuring the amount of tetrahydrobiopterin H4B present in a test sample is used to perform steps (a) and (b): (a) Contact a first test sample obtained from the subject at a first time point with a determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; (b) Contact a second test sample obtained from the subject at a second time point with a determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; Wherein monitoring the therapeutic efficacy of the subject's TAA further comprises step (c): comparing the measured amounts of tetrahydrobiopterin (H4B) present in the first test sample and the second test sample; Wherein a treatment is administered to the subject after the first time point and before the second time point, and wherein an increase in the amount of H4B present in the second test sample by at least 10% compared to the first test sample indicates effective treatment with TAA.

11. The use according to claim 10, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample comprises a high performance liquid chromatography (HPLC) column.

12. The use according to claim 10, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample comprises an immunoassay kit or a chemiluminescence assay kit.

13. The use according to claim 10, wherein an increase in the amount of H4B present in the second test sample by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% compared to the amount of H4B present in the first test sample indicates effective treatment with TAA.

14. The use according to claim 10, wherein the sample comprises plasma, serum or whole blood.

15. The use according to claim 10, wherein the determination device capable of measuring the amount of tetrahydrobiopterin H4B present in the test sample is further used to prescribe a modified TAA treatment for a subject in whom H4B has decreased or increased in the second test sample compared to the first test sample.

16. Use of a determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in a test sample for manufacturing a determination device for evaluating the severity of thoracic aortic aneurysm (TAA) in a subject, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample is used to perform steps (a) and (b): (a) Contact a test sample from the subject with a determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample; and (b) Measure the amount of tetrahydrobiopterin (H4B) present in the test sample; The further evaluation of the severity of the thoracic aortic aneurysm (TAA) in the subject further includes step (c): comparing the measured amount of H4B present in the test sample with the measured amount of H4B present in the standard; wherein the measured amount of H4B present in the standard is the measured amount of H4B in a healthy subject without TAA. Wherein a reduction of at least 10% in the measured amount of H4B present in the test sample as compared to the measured amount of H4B present in the standard indicates the severity of the subject's TAA.

17. The use according to claim 16, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample comprises a high performance liquid chromatography (HPLC) column.

18. The use according to claim 16, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample comprises an immunoassay kit or a chemiluminescence assay kit.

19. The use according to claim 16, wherein the sample comprises serum, plasma or whole blood.

20. The use according to claim 16, wherein the determination device capable of measuring the amount of tetrahydrobiopterin (H4B) present in the test sample is further used to prescribe TAA treatment for a subject with a reduction in H4B as compared to the measured amount of H4B present in the standard.

21. The use according to claim 16, wherein a reduction of at least 20%, 40%, 50%, 60% or 80% in the measured amount of H4B present in the test sample as compared to the measured amount of H4B present in the standard indicates severe TAA.

Images