CCTS: Pilot Grants

Lay summary:
Pathological cardiac hypertrophy (thickening of the heart muscle) underlying heart failure occurs as a maladaptive response to many forms of cardiac disease, including hypertension and myocardial infarction and is the single leading cause of death in the U.S (1). The high prevalence of pathological myocardial hypertrophy in cardiovascular disease coupled with a lack of effective treatment highlights a need for novel therapeutic treatments in this area. Thus, new insights into the molecular mechanisms involved in this disease process are key to the discovery and development of novel therapeutic agents for suppressing or reversing adverse cardiac growth. The focus of our application is to understand the signaling mechanisms of AKAP-Lbc-mediated pathological cardiac hypertrophy. Based on understanding of AKAP-Lbc scaffold function, we will address whether it is possible to prevent hypertrophic signaling. We propose that the scaffold protein A-Kinase Anchoring Protein (AKAP)-Lbc plays a central role in mediating pathological hypertrophic signaling. AKAP-Lbc nucleates a signaling complex composed of crucial proteins involved in the hypertrophic response, including protein kinase D (PKD). PKD is emerging as a key player in cardiac hypertrophic signaling and appears to have a pivotal role in the altered gene expression and cardiac remodeling seen in heart failure. Currently, little is known about PKD function and its regulation in the heart, however our supporting data suggests the importance of activation of PKD bound to AKAP-Lbc in mediating cardiac hypertrophy. To test the concept that AKAP-Lbc-bound PKD plays a role in the induction of pathological cardiac hypertrophy leading to heart failure, we have developed a transgenic mouse that expresses a form of AKAPLbc that cannot bind PKD. We will determine the cardiac function in AKAP-Lbc-ΔPKD mice using different models of human disease states, including pressure overload, chronic GPCR-agonist infusion and myocardial infarction to induce pathological cardiac hypertrophy and remodeling. Based on our supporting data we predict that abolishing AKAP-Lbc-anchored PKD signaling will diminish cardiac hypertrophy, remodeling and heart failure under these conditions. In summary, these proposed studies will rigorously define the in vivo role of AKAP-Lbc-anchored PKD in mediating cardiac hypertrophy in response to different modes of cardiac stress, thereby providing novel mechanistic information leading to new therapeutic strategies to counteract pathological hypertrophy.

Lay summary:
Angiogenesis, the growth of new blood vessels, and lymphangiogenesis, the growth of new lymphatic vessels, are primarily controlled by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). Specifically, VEGFR-2 has been implicated in the pathological processes of many conditions, including systemic sclerosis, non-small cell lung carcinoma, breast cancer, melanoma, and hematological malignancies. Furthermore, chronic rejection of transplanted human tissues, including corneas, is associated with lymphangiogenesis. Understanding the mechanisms of VEGFR-2 function may provide additional tools for manipulating the blood and lymphatic vascular systems and thus, lead to treatments for many of these conditions. The cornea, which is clear and avascular when healthy, is often used in angiogenesis and lymphangiogenesis research, but the results can also be extrapolated to other pathologies. Currently, anti-VEGF treatments focus on complete blockage of growth factors and their receptors; however, recent research indicates that there are distinct advantages to selective inhibition.
In this project, we will examine corneal angiogenesis and lymphangiogenesis and corneal transplant rejection in two different mouse models in which the VEGFR-2 gene is selectively knocked out in corneal epithelial cells and keratocytes or corneal vascular endothelial cells. Corneal epithelial cells and keratocytes contain soluble VEGFR-2 (sVEGFR-2), which is an anti-angiogenic, truncated form of membrane-bound VEGFR-2 that acts as an endogenous VEGF-C Trap. Albuquerque et al. recently showed that conditional deletion of VEGFR-2 in corneal epithelial cells and keratocytes deleted sVEGFR-2 and enhanced corneal lymphangiogenesis. We are generating conditional vascular VEGFR-2 lox knockout mice in which the VEGFR-2 gene will be selectively knocked out in corneal vascular endothelial cells. We are also generating Le-Cre-VEGFR-2 lox mice in which VEGFR-2 will be selectively knocked out in corneal epithelial cells and keratocytes. Our preliminary data indicate that targeted inhibition of tissue-specific VEGFR-2 may provide more effective control of angiogenesis and lymphangiogenesis. The proposed scientific aims will focus on delineating the functions of vascular VEGFR-2 in these processes. The long-term goal of this project is to use tissue-specific, conditional VEGFR-2 knockout mouse models to determine the most effective way to modulate VEGFR-2 to obtain optimum inhibition of corneal neovascularization. Based on our preliminary data, we hypothesize that tissue-specific inhibition of VEGFR-2 increases corneal angiogenesis and lymphangiogenesis and subsequently contributes to corneal transplant rejection.

Lay summary:
Pediatric bipolar disorder (PBD) is a major health concern with alarming rates of completed suicide, substance abuse, aggression and risk-taking behavior that makes it the toughest disorder to treat in child mental health. Therefore, understanding its underlying pathophysiology is crucial to facilitate early identification of this illness. In addition, disturbingly high rates of relapse and low rates of recovery of this disorder seek urgent attention to not only develop targeted treatments but to predict biomarkers of treatment response. Although the etiology of PBD remains to be poorly understood, bipolar disorder is conceptualized as genetically influenced disorders of synapses and circuits. MicroRNAs (miRNAs) are a prominent class of gene expression regulators that have critical roles in neural development, dendritic spine morphology, and circadian rhythm, processes hypothesized to be involved in pathophysiology of bipolar disorder. miRNAs are present not only in tissues, but also in various biological fluids including blood cells. Remarkably, miRNAs circulate in the peripheral blood in a highly stable, cell-free form that is protected from endogenous RNase activity. Interestingly, increasing evidence suggest that extracellular miRNAs can be uptaken by host cells, influencing their gene expression. It is, therefore, tempting to speculate that circulating miRNA may contribute to disease pathogenesis. The recent discovery that disease-specific miRNAs are present in body fluids has opened up the possibility of using them as non-invasive biomarkers for early detection, classification and treatment prediction. In fact, a recent study suggests that miRNA-134 is differentially affected in plasma of bipolar patients before and after treatment. This miRNA is known to be associated with BDNF, which we have found to be less expressed in blood cells of PBD patients. In this pilot-project proposal, we plan to focus on setting the stage so that measurements of plasma miRNAs and other small RNAs can be properly interpreted in terms of the underlying methodology as well as their biological context in terms of their use as biomarkers for diagnosis, treatment response, direct response to drug administration, or expression of symptomatology in PBD patients. In Aim 1, we will optimize methods of fractionating plasma (e.g. isolating exosomes, microvesicles, and non-vesicular supernatant), isolating total RNA, and measuring miRNAs using RT-PCR and Illumina deep sequencing platforms. In Aim 2, we will profile the expression of known miRNAs in plasma obtained from 20 PBD patients taken before vs. after 8 week lithium treatment and 20 age- and gender- matched control subjects. The data will also be assessed in light of drug levels in blood and symptom rating scales taken before and after treatment, to assess the relation of microRNA levels to drug levels, extent of response to treatment and presence of manic or depressive symptoms at the time of blood draw. We will also utilize cognitive dysfunctions measured in these patients as a function of miRNA changes. Finally, in Aim 3, PBD vs. control subjects will be profiled using Illumina deep sequencing to identify possible changes in novel miRNAs and other small RNAs, as well as to assess possible differences that cannot be detected by RT-PCR (e.g., RNA editing and 5' or 3'-modifications). Changes in plasma miRNAs may reflect changes due to systemic stress as well as alterations in brain microRNA pathways. By identifying changes that have robust clinical correlates, we hope to find biomarkers that will inform better diagnosis, prognosis and treatment of PBD patients.

Lay summary:
A high level of glucose (sugar) in the blood is a sign of uncontrolled diabetes or prediabetes, which can cause damage to proteins and effect how they function in the body. In addition, high blood glucose causes metabolic stress which can generate free radicals and further damage these proteins. This process is called advanced glycosylation. If high blood glucose persists then complications from diabetes such as heart disease, nerve, eye and kidney disease can result in a decreased quality of life, or death. Little is known about how advanced glycosylation from high blood glucose affects the skeletal muscles which are a primary site of glucose metabolism. Recently, glycation endproducts were found to disrupt glucose metabolism in rodent skeletal muscles suggesting that these compounds play a role in the development of the events that cause prediabetes and the onset of type 2 diabetes. However, these glycation endproducts have never been mechanistically studied in the muscles of human subjects with type 2 diabetes. This is surprising given that skeletal muscle is sensitive to insulin, a hormone that allows glucose to be metabolized in the body. In addition, obesity is also associated with the development of type 2 diabetes, high blood glucose and the production of free radicals, all of which are known to cause the formation of glycation endproducts. The primary goal of this research is to explore the relationship between glycation endproducts in skeletal muscle and glucose metabolism in type 2 diabetic and control subjects. We will test our hypotheses by performing a procedure that will determine how sensitive the muscle is to insulin and by taking a biopsy of muscle tissue from the leg to examine the amount of glycation endproducts. The leg muscles are an attractive tissue to study because they are easy to access via biopsy, the procedure is relatively low risk and the muscle has a high rate of metabolism. We will study the proteins in the muscle to determine how advanced glycation has damaged proteins important to glucose metabolism. We expect that greater amounts of glycation endproducts found in skeletal muscles are related to poor glucose metabolism and that proteins important for glucose metabolism have been damaged by the glycation process. This research is important so that early treatments for diabetes such as, drugs that target the glycation endproducts or lifestyle interventions such as diet and exercise can be validated and incorporated into medical therapy to help prevent and treat the development of long-term complications associated with diabetes. This proposal is both innovative and clinically relevant and will utilize an interdisciplinary, translational approach involving key CCTS CORE resources. The data generated from this proposal will help the PI transition into a novel area of research and will provide the foundation for future extramural funding applications to achieve scientific independence.

Lay summary:
Non-alcoholic fatty liver disease (NAFLD) is associated with insulin resistance and therefore is frequently found in obese and diabetic patients. Histologically, NAFLD occurs as a spectrum from mild hepatic fat accumulation (steatosis) only, to non-alcoholic steatohepatitis (NASH) characterized by steatosis with hepatocellular ballooning, lobular inflammation and fibrosis which can progress to cirrhosis with complications of hepatic failure and hepatocellular carcinoma. However, not all patients with fatty liver develop NASH. Therefore it is clinically relevant to 1) identify those patients with fatty liver that will go on to develop NASH and 2) design treatments which will prevent the progression of the disease. In support of these efforts, over the past decade biomedical scientists have used high-fat OR high-carbohydrate diets to generate obesity and insulin resistance in rodent models to study the impact on hepatic function. Although these models do develop fatty liver, they do not develop NASH, which limits their utility as translational models. Interestingly, very recent reports demonstrate that male mice supplied with free access to a "fast food" diet (ie. high-fat PLUS high fructose) do develop NASH, however information is lacking on liver function preceding NASH. Therefore, the specific aims of the proposed pilot study are to 1) identify pathways which are altered during the transition from simple steatosis to NASH in the fast-food diet mouse model, 2) correlate changes observed in the fast-food mouse model to existing gene expression profiles of human liver biopsies, where a subset show signs of NASH and 3) determine how fructose vs. glucose differentially modifies liver function (we predict the incidence of NASH will be reduced in mice fed an isocaloric diet when fructose is replaced with glucose). The information gained will provide the necessary proof of principle that the fast-food mouse model accurately reflects the pathophysiology of NAFLD/NASH and identify novel pathways involved in disease initiation and progression which can be future targets to develop early diagnosis, as well as prevention and treatment strategies.

Lay summary:
Alzheimer's disease is a brain disorder characterized by massive and progressive loss of neurons in specific brain areas. Neuronal loss is manifested by cognitive deterioration and memory deficits. Familial forms of Alzheimer's disease are rare. More than 95% of the patients suffer from the late onset sporadic form of the disease for which a genetic cause is yet to be found. There is no cure for Alzheimer's disease, and clinical trials of medications developed so far failed. This is due, at least in part, to lack of faithful models of the disease. For example, mouse models generated so far failed to imitate the progressive neuronal loss in the brain. In addition, these mouse models express rare genetic forms of the disease. Thus, in order to develop an effective therapy for Alzheimer's disease, it is imperative to establish new disease models that would replicate it faithfully. The discovery that cells from the adult can be reprogrammed to an embryonic stage [termed induced pluripotent stem cells (iPS)], opened up new avenues for disease modeling. Thus, for example, skin cells can be taken from Alzheimer's disease patients and reprogrammed to be human neurons, providing us with the unique advantage of studying human neurons of Alzheimer's patients. A fascinating phenomenon is the association between Down syndrome and Alzheimer's disease. Down syndrome is a mental retardation disorder caused by abnormal number of chromosomes. Interestingly, one of the genes that causes familial Alzheimer's disease is present on that extra chromosome in Down syndrome. Individuals affected with Down syndrome exhibit enhanced aging and early onset of Alzheimer's disease. In spite of this intriguing link, the molecular mechanism underlying this association is unknown. In a preliminary set of experiments, we successfully generated iPS from skin derived from mouse models of familial Alzheimer's disease. Having the methodology for the generation of iPS optimized, we now propose the generation of iPS from Alzheimer's disease patients experiencing sporadic or familial Alzheimer's disease, as well as from individuals affected with Down syndrome. iPS will be subcategorized based on diagnosis, risk factors and age of onset. We will use these human iPS to unravel neuronal vulnerability and loss in Alzheimer's disease, a fundamental, currently unsolved question, that is a prerequisite for the development of a successful therapy. These experiments will provide crucial new experimental models for aging, Alzheimer's disease and Down syndrome, new insight concerning the molecular mechanisms underlying sporadic Alzheimer's disease, and provide a platform for iPS cell based-drug discovery and therapy.

Lay summary:
Human Augmentics (HA) is the field of study that employs information technologies to amplify human capabilities. This CCTS pilot project uses HA to help people monitor their health and to affect long-term decisions for their wellbeing. More specifically, HA is applied to inner-city African American adolescent high-risk asthma patients to encourage them to use their daily asthma medication. Asthma sensors are modified to wirelessly send medication use (time, location) and peak flow meter data (amount of air forcefully exhaled in one minute) to a mobile device (e.g., smartphone), and then wirelessly sent to a Health Cloud. This Cloud is a computer cluster that securely harnesses anonymized data, compares with data from larger population samples, and makes long-term health predictions. Results are transmitted back to the mobile device to a dashboard application ¬ an easy-to-understand display of information ¬ using ³persuasive visualizations² to inform and persuade individuals toward healthier lifestyles. Users are reminded to take medication, rewarded for taking medications, and alerted if they lag in compliance. This CCTS project tests the hypothesis that the use of HA to provide information in real time and in a personal, interactive, and engaging way can have a transformative impact on human behavior.