2020 Research Grants
Two-year grants, $100,000
Marfan syndrome is a highly variable disease cause by mutations that affect the protein, fibrillin-1; the major component of structures in our tissues called microfibrils. We still do not fully understand what determines the severity of Marfa in different patients. Neonatal Marfan syndrome is the most severe form of Marfan, with patients dying in infancy, and mutations that cause neonatal Marfan affect a region of the fibrillin-1 protein called the "neonatal region." Interestingly, mutations that cause congenital contractural arachnodactyly (Beal's syndrome) affect the same part of the related fibrillin-2 molecule. Comparing the effects of neonatal Marfan and classical Marfan mutations, we recently found that the neonatal region contains a site likely to be involved in assembling fibrillin-1 in our tissues. If true, this has important consequences for our understanding of how neonatal Marfan develops. This project aims to test the idea that the neonatal region is needed for fibrillin-1 to assemble. New technology will be applied to create cells that make only mutant fibrillin-1 molecules, allowing us to test their effects directly. We will also look at whether neonatal Marfan mutant molecules block the function of normal fibrillin-1 molecules and whether this is why they cause a more severe disease. In addition, we will create an assay to study how mutations affecting fibrillin-2 cause Beal's syndrome. By combining the information from these experiments, we can begin to build a picture of how these parts of the fibrillin molecules perform their functions. The results will be important not only in understanding how these diseases develop, they will also help us understand the structure and assembly of microfibrils and how they control the development and maintenance of our tissues. This information is vital for the development of new strategies to combat these diseases.
Patients affected by Marfan syndrome are prone to develop progressive aortic enlargement that finally leads to the formation of aortic aneurysm, commonly at the first tract of the aorta, near the heart. This vascular complication is essentially due to a defective protein, fibrillin 1, which determines the weakness of the aortic wall, which loses the necessary elasticity to burden blood pressure stress. Together with elasticity loss, the aortic wall of Marfan patients shows an increased stiffness mainly due to fibrosis. These negative features are regulated by many proteins, including EMMPRIN whose main role is to directly and indirectly regulate stiffness: the direct action of EMMPRIN is based on deposition of stiff proteins like collagen 1, while the indirect one consists ofn triggering other proteins to destroy structural wall components.
We have recently described for the first time that EMMPRIN is highly present in the aortic wall of Marfan aneurysms and, more importantly, that collagen 1 levels decrease by blocking EMMPRIN function. Moreover, we found that the partial lacking of EMMPRIN in a mouse model recapitulating Marfan syndrome determines a milder aortic dilatation over time. To comprehensively understand the role of EMMPRIN in Marfan aortic aneurysm development and progression, we will take advantage of genetic engineering to eliminate EMMPRIN in another type of mice recapitulating a more severe form of Marfan. In parallel, we will treat these Marfan mice, which are prone to develop aortic aneurysms at young ages, with a drug able to inhibit EMMPRIN activation. The aorta of all these mice will be monitored by echocardiography, as for patients in clinical practice, and the fibrosis of the aortic tissue of these mice will be evaluated by molecular biology techniques. The collected data will be useful to compare aortic dilatation extent in Marfan mice with and without EMMPRIN inhibition and will allow us to determine, at a preclinical level, the exact contribution of EMMPRIN to the development of Marfan aortic disease. Interestingly, since we found that EMMPRIN levels are modulated in the blood of Marfan patients and that these values are associated with the presence of aortic aneurysm, in Marfan mice we will evaluate whether soluble EMMPRIN levels change in parallel with aortic dilatation progression, and therefore if they could be used as predictive marker.
The importance of this study relies on the fact that, to date, beyond surgery, no specific pharmacological therapy can avoid or limit aortic aneurysms in Marfan patients. Moreover, the reduction of surgical procedures, hospitalizations and control visits will result in considerable fallouts in the public health field. The proposed project is, therefore, aimed to better understand a novel possible causative mechanism at the basis of aortic aneurysms in order to introduce a plasmatic marker to monitor Marfan thoracic aortic dilatation, as well as to pave the way for novel specific drugs targeting the aortic wall of Marfan patients. Noteworthy, specific EMMPRIN inhibitors are already available for patients, suitable for drug development in the future.
Vascular Ehlers-Danlos Syndrome (VEDS) is a genetic connective tissue disorder associated with fragile blood vessels and other distensible organs. VEDS is caused by mutations in collagen-III, a major structural component of the extracellular matrix (ECM) that provides strength and structure to blood vessels and other elastic tissues. Mutations in collagen-III causes weakening of the blood vessels and internal organs that leads to life-threatening bleeding and an increased risk of death due to rupture of blood vessels and organs at a young age. Current VEDS interventions are only aimed at alleviating symptoms with no cures.
Our research goal is to understand how specific mutations in collagen-III lead to altered blood vessel structure and/or cell behavior in VEDS. Dense, collagen tissues have rope-like fibers, and mutations in collagen-III can alter the strength of the individual fibrils of the fibers. However, VEDS mutations can also occur in cell-binding regions of collagen-III. We hypothesize that VEDS mutations occurring in these cell-binding sites may not only affect the triple helix structure but also interfere with the information cells receive from the fibers subsequent production of new extracellular matrix, which is crucial for maintaining the integrity of blood vessels.
To study the influence of these mutations, ideally sources of collagen-III would be readily available. However, patient-derived, mutated collagen-III is difficult to procure, and each patient may present with a different mutation, which makes a systematic study extremely difficult. Instead, we propose to use materials made in bacteria. Bacteria can produce collagen-like peptides (CLPs) that form the base triple-helical structure of collagen-III, and whose sequence can be easily modified to specifically include common mutations from VEDS. To study our hypothesis, we will make CLPs that include normal or mutated cell binding sequences. We will modify the CLPs to facilitate their formation into a hydrogel mesh of fibrils and test the properties of these meshes to understand how the mutations affect this inherent strength. We will then incubate smooth muscle cells within in the hydrogels in a three-dimensional culture system. The cells will attach to the CLPs and reorganize the gel into a vessel-like tissue by pulling and contracting the mesh and by synthesizing new extracellular matrix. We will then evaluate how different mutations affect the strength and composition of the engineered vessels. By studying how the mutations in collagen-III affects blood vessel strength, we hope to open understand the impact of specific mutations on the severity of VEDS and to open new therapeutic avenues for patients with VEDS, including potential targets for gene editing.
Thoracic aortic aneurysm (TAA) is a major cardiovascular health problem characterized by a dilated aorta that may eventually dissect or rupture. TAA is associated with Marfan syndrome and other connective tissue disorders caused by genetic mutations in extracellular matrix (ECM) proteins. The aortic wall in TAA is characterized by degraded and fragmented elastic fibers in the ECM, regardless of the underlying molecular cause, suggesting that targeting degraded elastic fibers may be appropriate for monitoring and treating TAA. Nanoparticles (NP) conjugated to an antibody that binds to degraded elastin have shown promise for diagnosis and treatment of abdominal aortic aneurysm (AAA) in acute, chemical models of the disease in animals. Gold NP bind in vitro and in vivo to AAA tissue and can be used as a contrast agent for micro-CT imaging to identify fragmented elastic fibers and associated aortic dilation. Albumin NP loaded with pentagalloyl glucose (PGG) protect elastic fibers and prevent or reverse AAA. PGG is the core structure of tannic acid and stabilizes elastic fibers, protecting them from degradation. We, and others, have shown that PGG has protective and restorative effects on the mechanical changes associated with elastic fiber degradation in arteries. Despite the promising data on NP use for imaging and treatment in AAA, they have not been used in TAA. As AAA and TAA have important similarities and differences, it is important to expand the previous AAA studies to TAA. We propose to use a mouse model of genetic TAA caused by a mutation in the fibulin-4 protein that is directly related to fragmented elastic fibers. The mice are a model of a human connective tissue disorder associated with TAA. Fragmented elastic fibers are only found in the large arteries, such as the aorta, in these mice, so our NP will only target these vessels. We will perform imaging studies with micro-CT to investigate the localization of gold NP with respect to TAA and fragmented elastic fibers. We hypothesize that due to increased fragmentation in the TAA and associated endothelial dysfunction, the gold NP will localize to the TAA and the NP concentration will correlate with TAA severity. We will also perform treatment studies with PGG loaded albumin NP to investigate the efficacy of PGG treatment in preventing progressive elastic fiber degradation and aneurysmal dilation in these mice. We hypothesize that PGG delivered by the NP will bind to the improperly assembled elastic fibers and prevent further degradation that occurs with normal aging in these mice and slow TAA progression. Our studies may lead to new imaging and therapeutic protocols that can be used to diagnose or treat TAA in Marfan syndrome and other connective tissue disorders.
Clinical Research Grant
Two-year grant, $100,000
Patients with Marfan syndrome exhibit cardiovascular- and musculoskeletal-based conditions due to abnormal function of an important gene that helps to create soft tissue within the heart and muscles. This abnormal tissue formation causes the heart and muscles to not function properly, which results in poor patient health, poor physical function and severe pain in patients with Marfan syndrome. Although there are treatments that exist for the cardiovascular-based conditions in Marfan syndrome, there is not much information on how the muscles around the joints work and potentially cause joint pain in patients with Marfan syndrome. Although patients with Marfan syndrome have weak quadriceps and hamstrings muscles, we do not have any knowledge on whether or not the muscles around the hip joint are affected by Marfan syndrome. Approximately 46% of patients with Marfan syndrome have hip joint pain and also exhibit an abnormally shaped hip joint. This combination of weak hip joint musculature and abnormal joint shape can lead to high amounts of hip joint loading during walking, which causes hip pain and the hip joint to break down, eventually leading to hip osteoarthritis.
Our project will determine how hip joint muscle weakness, size and amount of fat within the muscle affects how the hip joint moves during walking in patients with Marfan syndrome. We will use a combination of magnetic resonance imaging (MRI), gait analysis, and surveys to measure the strength, size, and amount of fat within the hip abductors (muscles that make the hip move side to side) and the corresponding effects on hip joint movement patterns, hip joint pain, and cartilage health. The results of this project will provide an understanding of how the hip joint functions during walking in patients with Marfan syndrome and will allow clinicians to develop treatments that can improve hip abductor function, reduce hip joint pain and reduce the risk of hip osteoarthritis in people with Marfan syndrome.
Early Investigator Grant
Two-year grant, $75,000
Marfan syndrome is a genetic disorder that affects the body’s connective tissue and occurs in 1:5000 individuals. Connective tissue holds all the body’s cells, organs and tissue together through proteins and other molecules. The protein that plays a role in Marfan syndrome is called fibrillin-1. Marfan syndrome is caused by a defect (or mutation) in the gene that tells the body how to make fibrillin-1. This causes problems in connective tissues throughout the body, which in turn create heart and vascular problems leading to thoracic aortic aneurysm with dissection (TAAD) and often death. TAAD is a common life threatening disorder and a major manifestation of Marfan. TAAD begins with expansion of the ascending aorta (the main blood vessel that carries blood away from the heart to the rest of the body) and ends with dissection or rupture of the vessel and sudden death. TAAD still accounts for > 8000 deaths annually due to ruptured TAA.
Due to major knowledge gaps in our knowledge about how TAAD starts and advances within the aorta, there is currently no dedicated drug-based therapy for TAAD. In spite of significant research effort, drugs for TAAD management are currently limited to anti-hypertensive drugs that delay but do not prevent or cure disease progression. Surgery is the only alternative to overcome or delay the burden of patients. Using an innovative research strategy, we have recently used a validated mouse model of the disease (Marfan mice) to link TAAD development with increased activity of an enzyme called Hipk2. Both human and mouse aortas express Hipk2 and both Marfan patients and Marfan mice have increased amounts of Hipk2 in their aortas. Importantly, we have also reported that Marfan mice with genetic inactivation of Hipk2 fail to develop TAAD, suggesting that we could prevent TAAD progression by blocking the way Hipk2 works in the body. Our preliminary data strongly suggest that Hipk2 plays a role, at least in part, at the epigenetic level [i.e., it modifies how genes are activated or repressed in a way that is independent of the DNA sequence]. Here, we propose to use animal and cellular studies to examine 1) which molecules of the epigenetic machinery respond to Hipk2 activation and 2) how they work together in the diseased aorta to advance TAAD. Thus, we hope to identify new factors of TAAD in Marfan that can be blocked collectively or separately using drugs that are already approved or can be defined. Together, these animal studies will provide evidence-based support for testing new compounds in clinical trials involving TAAD patients.
Victor A. McKusick Fellowship Grant
Two-year grant, $100,000
The aorta is the largest artery in the human body. Blood pumps from the heart into the aorta. From the aorta, blood travels to the rest of the body. When the walls of the aorta develop an enlargement or weakness (called an aneurysm), the aorta can tear or rupture, causing death unless immediate surgery is possible. Marfan syndrome is a genetic disorder that affects the body’s connective tissue. Aortic enlargement, one of the common Marfan features, can be life-threatening. Unfortunately, no medication is yet available that can prevent the development of aortic disease for Marfan syndrome patients.
Understanding the cells that make up the parts of our body, like the aorta, has helped scientists develop new kinds of treatments for many diseases. Different kinds of cells each have a role to play, but they also cooperate with each other. Cells are able to work together by communicating with each other in complex ways. Scientists call this communication “cell-to-cell crosstalk.” By learning how cells communicate, we can sometimes find problems that cause disease.This study is specifically looking at the ways two kinds of important cells in the aorta communicate with each other. Using recent advances in scientific understanding and technology, we are able to study how gene expression affects this communication. We will compare the communication between cells in normal aortas to the communication between cells in aortas from patients with Marfan syndrome. By finding the differences, we may discover an interaction between the cells that can be treated with a new medication or therapy.