Abstract:  

Abstract:  

Abstract:

Abstract:  

Abstract:  The vascular endothelial barrier is a crucial component in the immune response, acting both as a physical barrier that prevents foreign bodies from entering the tissue as well as a chemical barrier that mediates the flow of small molecules and leukocytes. It has been shown previously that the components of adherens junctions play important roles in the regulation of the endothelial barrier. Recent research in our lab has shown specifically that VE-cadherin phosphorylation patterns in response to Src activation correlate with the initial strengthening and subsequent weakening of the endothelial barrier, a finding that adds a new dynamic to the accepted idea that Src activation only weakens the endothelial barrier. Tyrosine 731 in VE-cadherin was shown to be required for Src-mediated strengthening of the barrier, and tyrosine 685 phosphorylation was shown to coincide with the internalization of VE cadherin and weakening of the barrier. Our lab has done affinity purification mass spec analysis of VE-cadherin binding partners to WT and Y731F mutant VE-cadherin after 30 minutes of Src activation. We have identified various binding partners whose binding is dependent on Y731 as well as on Src activation. We propose a series of experiments that will allow us to better understand the importance and role these binding partners have in the endothelial barrier dynamics observed previously in our lab.

Abstract:  The cell death program pyroptosis has been shown to play an important role in the development and homeostasis of multicellular organisms. It is also implicated in inflammatory response-derived damage to the pulmonary endothelium and cardiovascular tissues. While the formation of oligomeric membrane pores by gasdermins to execute cell lysis is well characterized, unlike other cell death pathways such as apoptosis and necroptosis, it is not always lethal. The molecular mechanisms that diminish pyroptotic lethality not only facilitate further cell biology in surviving cells but will inform further bioengineering of this family of proteins. This project aims to engineer optogenetic and biosensing gasdermin tools to provide a direct, exogenous, and non-inflammatory activation of gasdermins in live cells. Our efforts will build a first-in-class platform to precisely and specifically analyze gasdermin pore dynamics using a combination of electrophysiology, optogenetic design, live cell fluorescent biosensing and imaging analysis. Data from this project already established an optogenetic gasdermin D, and revealed that previously unknown gasdermin D dynamic is critical for controlling the progression of pyroptosis and inflammatory cytokine release. Furthermore, pore-mediated signaling can drive the activation of specific protein kinases. Optogenetic tuning can be shown to offer precise control over not only pyroptosis, but importantly kinase signaling and thus the cell biology endpoints of these kinases such as transcription factor activation. By uncovering the unknown role of these molecules besides innate immune defense, we reveal important cell biology and thus novel bioengineering opportunities of gasdermin proteins.

Abstract:  Nearly half of all heart failure cases are heart failure with preserved ejection fraction (HFpEF). While there are no differences in mortality rates across the spectrum of ejection fraction, it is only HFpEF that is without any beneficial therapeutics. This asymmetric response to drug treatments highlight the divergent pathological conditions between HF with reduced or preserved ejection fraction. Unique to the pathology of HFpEF, is the notion that cardiac dysfunction arises due to inflammation in non-cardiac tissues, especially the endothelium. Unsuccessful efforts to treat HFpEF reflect shortcomings in our understanding of this complex condition which is often associated with multiple comorbidities. To address this issue, we have implemented a two-hit model of HFpEF, combining HFD with L-name to induce metabolic and hypertensive stress to recapitulate patient populations more accurately. In performing an integrative analysis linking chromatin modifications with altered gene expression and proteomic signatures we intend to offer a much-needed resource that may assist in multidisciplinary efforts to discover new and underappreciated pathological mechanism. Among those underappreciated pathways we have identified a dysregulated gene across multiple human and murine RNA-seq data sets, that is known to be involved in mediating vascular tension and angiogenic activities and whose role in HFpEF has not yet been described.

Abstract:  Healthy hematopoietic stem cells (HSC) are maintained in a highly specialized microenvironment within the bone marrow, commonly referred to as the stem cell niche. Understanding the mechanisms by which the HSC niche regulates leukemia initiating cells, also referred to as leukemia stem cells (LSC), is crucial to improving treatment outcomes and creating new therapies for acute myelogenous leukemia (AML). In AML, expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity in affected individuals. The high overall relapse rates in AML suggest that persistent LSC are not targeted by currently used treatments. Although it is likely that the bone marrow microenvironment regulates LSC quiescence and proliferation, it remains unclear whether LSCs and healthy HSCs share the same niche or the extent to which this niche is remodeled to provide preferential support of malignant cells. Our previous work has shown that bone marrow megakaryocytes directly regulate HSC quiescence via platelet factor 4 (PF4), a niche chemokine highly and selectively expressed by megakaryocytes. Interestingly, recent studies also identified the lung as a novel potential niche for healthy megakaryocyte and hematopoietic stem and progenitor cells, although it is unclear if this microenvironment further provides support to LSCs. Here, we intend to investigate the role of PF4 and other megakaryocyte-derived factors on LSC proliferation and overall AML progression. It is our hope that this will also give insight into the extent to which AML remodels the megakaryocytic niche in the bone marrow and lung to become more permissive to LSC and bulk AML cells.