2013 Pilot Funded Projects
Bogi Andersen, MD
"Cell type-specific analysis of epigenetic marks in human tissues"
AbstractEpigenetic mechanisms are thought to account for altered gene expression in many human diseases, such as cancers and inflammatory skin diseases. Epigenetic modulations may also play a role in aging. Epigenetic mechanisms areof great interest in a number of diseases as they can account for environmental influences in disease. Consequently, there is great need to invent methods that can define the epigenetic landscape in specific cell types within normal and diseased tissues. So far the analysis of global histone modification by ChIP-seq has been limited to in vitro cultured cells or whole tissues which contain a complicated mixture of different cells each likely to contain different epigenetic landmarks. Alternatively, cells have been sorted based on cell surface markers and the lengthy cell isolation method is likely to leadto changes in the epigenetic landscape. We propose to develop a novel technique to isolate marker-specific cells fromepidermis by immunofluorescence sorting of nuclei from fixed tissues. We also propose to adapt novel methods that allowthe use of minimal amount of chromatin DNA for ChIP-seq. If successful, this approach, which is loosely based onmethods so far only used in Drosophila, would have strong
Jaime Landman, MD
Quantitative assessment of the effects of Hydrogen Sulfide (H2S) in renal ischemic and reperfusion injury for partial nephrectomy in a porcine model using Spatial Frequency Domain Imaging (SFDI)
The overall goal of our study is to understand the pathophysiology of renal ischemia and reperfusion injury (IRI) due to renal vessel clamping during partial nephrectomy. It is well established that decreased kidney function and chronic kidney disease (CKD) is associated with increased rates of death, cardiovascular events, hospitalization and significant medical costs. Mortality rates remain above 20 % per year with the use of dialysis and the annual direct medical costs for end-stage renal disease (ESRD) are nearly $23 billion1. Thus, it is critical that we preserve maximal kidney function and minimize IRI to prevent development of CKD/ESRD during partial nephrectomy. In order to study the effects of IRI we are employing a novel technology called Spatial Frequency Domain Imaging (SFDI) to measure the acute changes that occur during renal clamping and unclamping. One of the aims of this project is to demonstrate the application of SFDI during surgery as an accurate real-time monitor that is able to indicate the development of irreversible ischemic renal injury. SFDI is a non-contact optical imaging system which has the ability to rapidly render quantitative 2-D wide-field maps of in-vivo tissue oxy and deoxy hemoglobin (Hb), total Hb and O2saturation. SFDI has been previously validated in porcine ischemic tissue flap models and recently translated to human studies for skin flap oxygenation for breast reconstruction after mastectomy2. The most updated version of SFDI is designed for rapid imaging capable of measuring absorption and scattering maps using four LED wavelengths and a camera readout time < 1second. By measuring these parameters real-time, the surgeon will be able to detect the progression of IRI, in which intra-operative modifications can be made to prevent/minimize renal injury. We also plan to correlate these changes demonstrated on SFDI with histopathology & immune-histochemistry (IHC) for signs of apoptosis, tubular injury and also measure the different biomarkers of oxidative stress and renal injury (TNF-alpha, TLR-4, NGAL, KIM-1 and NHE-3).
For the final component of this study, we are planning to introduce a recently emerging gasotransmitter Hydrogen Sulfide (H2S) to investigate its protective effects on IRI and cellular injury. Our objective for this part of this study is to directly inject the kidney with H2S just prior to clamping to demonstrate the benefits against IRI. Recently, a wide range of studies in different organ systems (heart, liver and kidney) have been published on the protective effects of H2S against IRI as a mediator of inflammation, inhibitor of cellular respiration and cytoprotective and antioxidant effects. Our study will be an acute and chronic kidney model, which has not been previously reported in the literature. Moreover, a porcine model (which closely resembles human anatomy) has not been utilized to understand the potential role of H2S in IRI. We believe that H2S can play an important role against IRI and prevent chronic irreversible damage during partial nephrectomy. The overall effects of H2S will be studied in conjunction with SFDI, histopathology & IHC and biomarkers of renal injury as mentioned above.
Ellis Levin, MD and Young Jik Kwon, PhD
Reversing Tanoxifen Resistance in Breast Cancer
Acquired resistance against breast cancer therapy (e.g., Tamoxifen, TAM) is a keychallenge in the clinic and greatly affects clinical outcome. We recently discovered thatTAM-resistance in breast cancer occurs when TAM binds to the mitochondrial estrogenreceptors (ER)-β as an agonist (like estrogen), resulting in low formation of reactiveoxygen species (ROS) and the growth and survival of breast cancer cells. Therefore,inhibiting the process downstream of mitochondrial ER is a promising strategy toovercoming TAM-resistance in breast cancer. This proposed study hypothesizes thatTAM-resistance in breast cancer can be efficiently reversed by delivering smallnucleotides, such as small interfering RNA (siRNA), against an ER pathway thatconveys resistance. The object is to restore high amounts of cytotoxic ROS formationthat is characteristic of tumor sensitivity to TAM, using TAM and stimuli-responsivenanoparticles. The nanoparticles are engineered to deliver siRNA against the ROSquenchingenzyme, MnSOD, to keep ROS high to kill the cancer cells, in the setting ofTAM. Thus, we will restore TAM sensitivity. The proposed research specifically aims 1)to establish optimal nanoparticle-mediated reversal of de novo TAM-resistance in vivofor breast cancer and 2) to induce breast tumor apoptosis by restoring the cytotoxicresponse to the selective estrogen receptor modulator (SERM), thus reversing acquiredTAM-resistance by targeted nano-siRNA delivery. The significance and innovation ofthe proposed study are 1) addressing a major clinical challenge in breast cancertherapy (drug-resistance), 2) translating the discovery of a key target for therapy, and 3)novel approach to overcoming breast cancer drug-resistance using a novelnanotechnology tool. Successful completion of the proposed research will develop safeand effective therapy for drug-resistant breast cancer.
Roxanne Silver, PhD
A Gene-Environment Study of Coping among an Indonesian Sample Exposed to Repeated Natural Disasters
In recent years, evidence has emerged identifying specific genotypes that influence an individual’s response to traumatic life events (Amstadter et al., 2009). Recent advances in molecular genetics suggest that genes regulating neurohormonal pathways in endogenous cannabinoid system (FAAH, CNR1), the HPA axis (glucocorticoid; FKBP5, RGS2), frontal-limbic (serotonin and dopamine; DRD2, 5-HTTLPR), and noradrenergic (norepinephrine; ADRB1) systems are linked to the risk of the development of posttraumatic stress disorder (PTSD) and other comorbid psychiatric disorders (e.g., depression, anxiety). However, the role of genes in the stress response is complex – genes interact with environmental events to moderate both the susceptibility of individuals to PTSD and the potential efficacy of psychological interventions. Although family environment appears to shape genetic susceptibility to trauma, there is a surprising lack of research addressing the role of genetics in helping families cope. Thus, there is a great need for studies that can identify gene-gene and gene-environment interactions that affect resilience and susceptibility to PTSD in families following collective trauma. Such studies could provide invaluable information for the development of interventions (e.g., social, pharmacologic, behavioral) tailored to meet the needs of individuals and their families. We designed and conducted a study in collaboration with Universitas Sanata Dharma in Yogyakarta, Indonesia to investigate how genetic susceptibility and gene by environment (G X E) interactions moderate disaster responses in a chronically exposed population. The nation of Indonesia is located within a geological area of instability, subjecting its inhabitants to more than 55 disasters including major earthquakes, floods and landslides, volcanic eruptions and disastrous tsunamis since 2000. As such, it is an ideal location to examine how humans react mentally and physically to repeated natural disasters. This research effort employed a cross-cultural validation procedure of all psychosocial measures and three waves of data collection on 428 parents and 545 children from six elementary schools in the rural regions near Yogyakarta. A salivary sample to assess genetic markers was collected on ~90% of the parent sample (N=383) and ~60% of the children (N=316), representing 72% of the full sample. The specific aims of this proposal are to examine how social and community environments interact with DRD2, FKBP5, 5-HTTLPR, and the endocannabinoid-regulating genes to affect the mental health of a population subjected to chronic and repeated exposure to disasters. Findings from this pilot study will be used to design and conduct future work to examine whether certain individuals may be more responsive to trauma-based interventions than others. To our knowledge, this is one of the first comprehensive projects using a biopsychosocial model to examine the effects of repeated, chronic exposure to trauma; it has the potential to make several important contributions. First, it may help clarify the role of these genes in response to multiple traumas. Second, we will examine gene-gene and gene-environment interactions that can inform development of person-specific interventions that fit the individual, familial, and community needs after repeated trauma. Because we have included parent-child dyads, we can examine how intergenerational patterns of parent-child genotype interactions affect mental health, and how the social environment helps shape parent-child dyadic responses to repeated exposures. Such information would be extremely valuable for developing family- and community-centered interventions to help children and families cope with their experiences.
Koyoko Yokomori, PhD
Development of a novel ChIP-based diagnostic assay for FSHD
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies and ischaracterized by progressive weakness and atrophy of facial, shoulder, and upper arm musculature, which cansubsequently involve the abdominal and foot-extensor muscles. Most cases (>95 %) of FSHD involve monoallelicdeletion of D4Z4 macrosatellite repeat sequences at the subtelomeric region of chromosome 4q(FSHD1). There are between one and ten repeats in the contracted 4qter allele in FSHD1 patient cells, incontrast to 11~150 copies in normal cells. In addition, ~5 % of FSHD cases are not associated with D4Z4repeat contraction (FSHD2), and their etiology remains undefined. Although FSHD is reported to have a one in20,000 incidence, there is great concern that the actual number of affected individuals is significantly higherdue to undiagnosed cases (with a likely incidence of 1/7,000). Proper diagnosis depends initially onrecognition of clinical signs and symptoms and differentiation of FSHD cases from other muscular dystrophies.Molecular studies have been used to reinforce the clinical impression. The primary approach has been throughdetection of 4qD4Z4 repeat contraction by pulsed-field gel electrophoresis (PFGE) following restrictiondigestion. However, this method cannot identify phenotypic FSHD (with no repeat contraction), and certainband patterns can prove difficult to interpret. More recently, DNA hypomethylation at the D4Z4 locus was alsofound to serve as a diagnostic marker. However, severe DNA hypomethylation was also found in cells frompatients suffering from the unrelated “immunodeficiency, centromeric instability and facial anomalies” (ICF)syndrome, and thus is not FSHD-specific. We discovered a specific change in histone modification (histoneH3 lysine 9 trimethylation (H3K9me3)) at the D4Z4 repeat sequences that is detected in both FSHD1 andFSHD2 patient cells. Importantly, this change is highly specific for FSHD; no significant change of H3K9me3was observed in limb-girdle muscular dystrophy (LGMD), oculopharyngeal muscular dystrophy (OPMD), orinclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD),which are clinically similar and often misdiagnosed, as well as Duchenne muscular dystrophy (DMD) and ICFsyndrome patient cells. Importantly, this change is seen not only in affected muscle cells, but also in patientfibroblasts from skin biopsies and lymphoblasts from blood samples. This indicates that the loss of H3K9me3is not an epiphenomenon of dystrophic muscle, but is indeed a marker specifically associated with both typesof FSHD that appears to occur early in development and is therefore found in many cell types of an affectedindividual. Thus, in this pilot project, we plan to test the possibility that ChIP can be used to detect the loss ofH3K9me3 in patient chromatin as a diagnostic method for FSHD. We plan to use peripheral bloodmononucleocytes (PBMC) from patient blood samples that can be obtained significantly less invasively (andless painfully) than standard muscle biopsy samples. Detection of H3K9me3 loss will be assessed bychromatin immunoprecipitation (ChIP) analysis. One problem is that the quality of commercially availableH3K9me3 antibodies can be variable and sometimes not sufficiently specific, which complicates evaluation ofthe results. Thus, we plan to improve the standardization of the ChIP assay by using the recombinant Fabfragment of an antibody proven to be highly specific for H3K9me3, effectively eliminating the issue of antibodyvariability. Specifically, we plan to develop a diagnostic ChIP assay by (1) titrating the recombinant Fabfragment and chromatin samples to define the ideal quantities of each, (2) determining proper blood storageconditions and the optimal method for chromatin isolation from PBMCs suitable for ChIP, and (3) modifying theChIP protocol to be suitable for a reproducible diagnostic application. We plan to assess the specificity of ourprotocol by testing blood samples from healthy members of patients’ families, from patients of different agesand disease severities, and from individuals with unrelated muscular dystrophies or unrelated diseases.
Hiroshi Yoshioka, MD
Study of knee cartilage degeneration by novel orientation/thickness dependent T2 and T1rho mapping approach
Osteoarthritis is one of the most prevalent disorders in today’s society, resulting in significant socio-economic costs and morbidity. A host of new and exciting therapeutic modalities are being developed for the treatment of osteoarthritis, which include new chondroprotective and chondroregenerative drugs, osteochondral autografting, and autologous chondrocyte implantation. Therefore, it is important to detect early cartilage degeneration and understand its natural progression for treatment of osteoarthritis. MRI plays an important role in the assessment of internal derangement of joints and has been reported to be an excellent modality to diagnose cartilage lesions. T2 relaxation time and T1rho relaxation time have recently been used in physiologic and quantitative MRI evaluation of articular cartilage, associated with matrix damage, particularly loss of collagen and proteoglycan integrity. However, T2 relaxation time of normal knee cartilage is dependent on orientation to the static magnetic field as well as on different cartilage layers due to relationship between the magic angle effect and collagen orientation. This causes orientation/thickness dependent normal variation of cartilage T2 relaxation time in the knee. In this project, we hypothesize that we can evaluate early cartilage degeneration of the knee more effectively and accurately using a novel orientation/thickness dependent T2 mapping and T1rho mapping approach. First, we will recruit healthy volunteers without history of knee injury and scan their knees using T2 and T1rho map sequences. Orientation/thickness dependent T2 and T1rho relaxation times will be calculated using custom software. This will allow us to standardize normal T2 and T1rho profiles and evaluate subtle T2 and T1rho abnormality, i.e. early cartilage degeneration, in various locations on the femoral condyle accurately. Next, we will recruit patients with knee osteoarthritis and scan their knees with established T2 and T1rho map sequences. We will perform the same analysis and create orientation/thickness dependent T2 and T1rho mapping from them. Deviation of T2 and/or T1rho relaxation time from normal control T2/T1rho relaxation time will be diagnosed as cartilage lesions. This project will be collaborated with radiologist, orthopedic surgeon, and MR physicist at UCI.