Age-related cardiac pathologies are currently endemic in most developed nations and account for a large number of premature deaths and huge medical expenditures. Although lifestyle-associated risk factors have frequently been correlated with risk of cardiac impairment, it is also clear that aging impairs heart function even in the absence of these risk factors. Caloric restriction (CR) is known to extend lifespan and offset age-related impairment in diastolic function in murine models. Rapamycin is a CR mimetic that also extends lifespan (when given throughout life) and is protective in models of cardiac hypertrophy and failure. However, the potential role of Rapamycin for treatment of impaired heart function in aging has not been established. To determine the effect of short-term Rapamycin treatment on cardiac aging, we monitored cardiac function in 25 months old C57BL6/CR mice fed for 10 weeks with a control diet or a diet containing 14ppm encapsulated Rapamycin. Analysis by tissue dropper imaging showed improvement in diastolic function in the treatment group as early as 4 weeks. Normalized heart weights were also lower in the treatment group compared to old control mice, suggesting that short-term sub-acute Rapamycin treatment can reverse cardiac aging and regress age-related cardiac hypertrophy. Proteomic analysis showed increased stability (half-life) of the cardiac global proteome, increased abundance of mitochondrial proteins, and decreased oxidative damage of proteins in the treatment group. Further analysis revealed metabolic remodeling in the glycolytic and TCA pathways that improve energy metabolism and cardiac function in 10 weeks Rapamycin treated mice. Data from murine models offer potent approaches that may be replicable in humans and may prove effective in management of cardiac and perhaps other age-related ailments. In this project, we both assist in preparing the control and treatment diets, mice handling, imaging preparation, tissues harvesting, data analysis, and experimental design for future experiments.

Thrombotic microangiopathy is featured by endothelial activation and microvascular thrombosis, and is associated with a group of life-threatening disorders, such as thrombotic thrombocytopenia purpura (TTP), sepsis, and other small vascular diseases. With systemic inflammation, the vessels are often found to be obstructed by platelet rich thrombi, leading to tissue ischemia and organ failure. Understanding the mechanism behind endothelial activation and induced thrombi formation during inflammation has important clinical implications and can lead to therapeutic development. Here, I developed a novel in vitro microvascular platform that mimics the in vivo vascular architecture and function. This platform is generated based on a biocompatible polymer, polydimethylsiloxane (PDMS), using a soft lithographic method. A vascular network is fabricated in the PDMS mold, which is subsequently sealed with a glass coverslip to create an enclosed network. The device is lined with an extracellular matrix using type I collagen for structural support. Optimization was made to provide consistent, high-throughput testing by controlling the diameter and integrity of the vessel lumen. Human umbilical vein endothelial cells (HUVECs) were seeded into this device and cultured for 7 days. To recapitulate the inflammation during diseases such as sepsis, lipopolysaccharide was used and perfused through the vessels to stimulate the endothelium. Granules or secretory vesicles carrying von Willebrand Factor (VWF) were observed to release its contents from the activated endothelium, forming strands of VWF along the direction of flow. On-going studies include examining changes in the transcription factors during endothelial cell activation, cleavage and self-association of VWF strands under flow, and drug screenings to prevent VWF secretion and strand formation. The study is expected to not only elucidate the fundamental mechanism of microvascular diseases, but to also provide a base platform to develop preventative or therapeutic medicine for those affected by these diseases.

Today, approximately 13% of the world's population is affected by chronic kidney disease, with kidney transplantation being the only functional restorative treatment. However, the significant shortage of donor kidneys leaves 100,000 Americans with limited treatment options. My project seeks a solution to generate proper human kidney extracellular matrices through decellularization and subsequent recellularization to recreate functional kidney peritubular filtration unit for the application toward therapeutic development. The efficacies of cellular material removal using sodium dodecyl sulfate (SDS) and Triton X-100 were evaluated on porcine kidney sections. SDS solution showed better cellular removal and matrix preservation. To optimize organ decellularization protocol, decellularizations on organ porcine kidneys were performed using a pumping system that perfused SDS into the renal arteries of the kidneys. Decellularized organs retained intact microarchitecture and the essential extracellular matrix components such as laminin, fibronectin, and collagen IV. Future work will be focused on human kidney organ decellularization using the protocol previously developed and decellularized scaffolds repopulation, testing the cellularity and regenerated kidney functions. If successful, this project will not only lead to recreate human kidney peritubular filtration unit in vitro for toxicity testing and therapeutic development, but also provide insights about how the microenvironment controls the structure and function of the cells and tissues.

The incidence of esophageal adenocarcinoma (EAC) is increasing world-wide. Early and specific detection of EAC and its precursor non-cancerous stages, particularly Barrett’s esophagus (BE), is crucial to treatment and long-term survival. Currently, endoscopy coupled with serial biopsy is the gold standard for diagnosis. Standard endoscopy lacks specificity. BE presents as a salmon-reddish discoloration against the pale-pink color of healthy esophageal tissue. Cardia intestinal metaplasia (CIM) arising from gastric tissue is visually similar; both develop in response to prolonged acid reflux and are often confused during histological analysis. However, CIM is unrelated or even negatively correlated to EAC development. Distinguishing between BE and CIM at the gastroesophageal junction is crucial to minimizing the use of invasive biopsy. Targeting molecular markers specific to disease state can enhance specificity. Fluorescent dyes attached to targeting molecules are used to label these biomarkers. Instrumentation designed for in-vivo fluorescent imaging, like the ultrathin, flexible, multimodal scanning fiber endoscope (SFE) can then excite and image the fluorescent dyes, effectively red-flagging abnormal regions for both enhanced biopsy capture and point-of-care diagnosis. Reliance upon one biomarker alone is ineffective due to patient heterogeneity, as expression varies between patients due to factors including genetic background and lifestyle. Multiple biomarkers are necessary for specificity. The purpose of our work is to first demonstrate that the three cell surface biomarkers, EGFR, C-MET, and ERBB2, well established in other cancers, are appropriate for early EAC or BE diagnosis. We plan to demonstrate the simultaneous application of all three biomarkers to provide added specificity and sensitivity for diagnosis and ultimately prognosis. These goals will be achieved through immunofluorescent staining of cell lines and paraffinized tissue samples excised from patients. The efficacy of using multiple markers will be assessed through image processing and statistical analysis.