Zhenyu Li, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project 4: Platelet Activation with Obesity Promotes Atherothrombotic Vascular Events Zhenyu Li Obesity is associated with an increased risk of cardiovascular death independent of its other recognized consequences such as hypertension and hyperlipidemia. One potential contributing factor to this excess in cardiovascular deaths may be related to the pro-thrombotic and pro-inflammatory states induced by increases in adipose mass, both of which are critical components of the pathogenesis of the clinic manifestations of atherosclerosis. Platelets play a central role in arterial thrombosis, are activated in inflammatory states, and are directly influenced by specific adipokines, and therefore have the potential to serve as an essential mediator of the cardiovascular consequences of obesity. Consistent with this, obesity has been associated with increases in platelet aggregation, elevations in surface expression of markers of platelet activation such as P-selectin, and heightened platelet microparticle formation. More importantly, reduction in adipose mass leads to normalization of markers of enhanced platelet activation. However, how platelets are activated in obesity, and a causal role for platelet hyperactivation in obesity-related cardiovascular disorders remains to be established. Several characteristics and proven biological activities of platelets make them an appealing candidate for triggering and maintaining the inflammatory response of obesity. This study will test the central hypothesis that platelet activation/secretion secondary to obesity plays a causal role in triggering and maintaining the pro-inflammatory and pro-thrombotic state of obesity, creating a feedback loop involving adipose tissue, activated platelets and vascular endothelium that culminates in an environment favorable for atherothrombotic vascular events.

This research involves design and building of a three-dimensional (3D) microarray device, with position-controlled microspheres, to perform simultaneous, efficient, and accurate screening of complementary DNAs, RNAs, and protein receptors on a single platform. This new device is portable, self-contained, automatic, and cost effective. Applications of this device include medical screening, drug discovery, and gene sequencing. In particular, it performs inexpensive disease diagnosis and provide insight into the molecular basis in different patients.

In existing 3D microarrays, microspheres are placed randomly within a substrate. This random placement of the microspheres makes their packing inefficient and their data processing complex. To overcome these drawbacks, the investigators design and build new microarrays with position-controlled microspheres. They analyze the statistical accuracy in estimating the target concentrations by computing performance bounds, and apply the results to select the minimal distance between the microspheres and the best operating temperature, while ensuring desired optimal estimation accuracy. The minimal microsphere distance enables high packing, and the optimal temperature reduces the cost. The investigators implement the position-controlled microarray using a microfluidic approach; particularly, they emplace the microspheres using a hydrodynamic trapping mechanism and using on-chip microvalves and pumps. The long-term goal is to integrate this device with image sensors, electronics, and optofluidic imaging to build a complete lab-on-a-chip system.

DESCRIPTION (provided by applicant): Platelets are central to hemostasis because they respond to vascular damage and secrete a variety of granule cargo molecules, which are critical to thrombosis and its sequellae. Platelet secretion is a multistep process involving centralization of cytoplasmic organelles that provides a contractile force, membrane fusion controlled by membrane traffic proteins, and release of granule contents. The platelet integrin, ?llbß3 interacts with adhesive ligands such as fibrinogen and fibrin and mediates platelet adhesion and aggregation and thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. The overall goal of this proposal is to establish a link between the membrane traffic proteins and integrins that involve the two key events to platelet function, platelet cargo release and integrin activation. VPS33B is a member of the Sec1/Munc18 protein family that has multiple roles in exocytosis. VPS33B is defective in patients with arthrogryposis, renal dysfunction, cholestasis (ARC) syndrome. Platelets from these patients lack ?-granules. In an attempt to define the roles of ?-granules in platelet functio and thrombosis, we produced a mouse model of ?-granule deficiency, in which VPS33B was deleted only in megakaryocytes and platelets. In Specific Aim 1, we will determine the roles of VPS33B in ?llbß3-dependnet endocytosis and ?llbß3 outside-in signaling. ?-granule contents vWF and fibrinogen are significantly reduced in the platelets lacking VPS33B. Surprisingly, VPS33B-/- platelets fail to retract a clot and are defective in spreading on fibrinogen. Using the CHO recombinant ?llbß3 activation model, we showed that overexpression of VPS33B markedly potentiates cell spreading on fibrinogen and actin polymerization. Thus, we hypothesize that in platelets, there exists a crosstalk between the membrane traffic proteins and integrin activation and that VPS33B is a key contributor to ?llbß3 outside-in signaling. This hypothesis will be tested using the platelet- specific VPS33B conditional knockout mice and other VPS33B knockout mice such as whole-body knockout mice and a Chinese Hamster Ovary (CHO) integrin activation model. Our preliminary data demonstrated that VPS33B co-localizes with ?llbß3 as detected by confocal microscopy and forms a complex with ?llbß3 as detected by co-immunoprecipitation. In Specific Aim 2 we will use multiple approaches to characterize the binding determinants in VPS33B and the ß3 cytoplasmic domains for each other. In Specific Aim 3 we will identify the molecular mechanisms of VPS33B-dependent ?llbß3 outside-in signaling. Completion of these studies will not only identify a novel ?llbß3 binding partner that plays an important role in ?llbß3 outside-in signaling, but also for the first time establish a lin between membrane traffic proteins and integrin activation in platelets.

? DESCRIPTION (provided by applicant): Asthma is a common chronic inflammatory disease of the airways, which affects approximately 26 million people in 2010 and costs $56 billion each year in the US. In particular, 7 million children suffer from asthma (~9.5%) which causes more than 14 million lost school days due to asthma exacerbations. Although triggers to pediatric asthma exacerbations are well recognized such as infections, allergy, smoke, chemicals, and exercises, how the interactions among the environmental factors and the patients' biological and behavioral characteristics determine the susceptibility to and timing of such events is less clear. One significant barrier to the causal understanding is the lack of objective measures on exposure metrics correlated with patient physiological responses and activities. Therefore, there is a significant unmet need to develop integrated sensor monitoring systems that can be deployed in a child's daily life and collect real-life environmental, physiological and behavioral data for epidemiological studies of asthma. This work proposes to capitalize on the recent technological advancements in wearable sensors and microfluidic point-of-care diagnostics, and the ubiquitous smartphone platforms to develop novel ambulatory integrated sensor systems for collecting such real-life data, which will enable the rigorous testing of hypotheses on environmental contributions to the causation of asthma exacerbations. Specifically, we will utilize flexible electronics, handheld microfluidics and commercially available gas, flow and activity sensors to build a wristband monitoring environmental air pollution and patient activities a handheld immunosensor for saliva total IgE and inflammatory marker cytokine IL-13 tests, and a smartphone attachable spirometer. Three specific aims are proposed to achieve the sensor developments, integration with informatics platforms and validation with pilot studies on pediatric asthma patients. Specific Aim 1. Is to design and implement three ambulatory sensors: a wristband capable of monitoring air pollutions and patient activities, a handheld microfluidic immunosensor for total IgE and cytokine tests, and a smartphone attached spirometer for lung function measurements. Specific Aim 2. Is to integrate the ambulatory sensor array with smartphone based informatics platform that will developed by the companion PRISMS FOA RFA-EB-15-003. Specific Aim 3. Is to validate the ambulatory sensor arrays in a pilot clinical study with inner-city pediatric asthma patients.

Predisposition to disease and reaction to drugs only partially depend on our genetic code. The organization, packing and subtle dynamic interactions of the DNA in the cell nucleus can profoundly modify gene expression and affect human health. The DNA packing and dynamics can be altered by factors and molecules known as epigenetic modifiers of gene expression and are of great interest in oncology and cardiology. The overall goal in this EFRI project is to obtain better understanding of the action of the dynamic chromatin modifiers in heart cells to inspire new therapies at the intersection of oncology and cardiology and help develop safer and more effective drugs. The team will deploy new optical techniques and light-sensitive tools to address this challenge.

Histone deacetylase inhibitors (HDACi) represent a class of powerful chromatin modifiers. Active development of HDACi as therapeutic agents has yielded over 500 small molecules, currently at different stages of clinical trials. The team will leverage a key feature of HDAC-mediated control of the chromatin structure, namely its fast dynamics and reversibility compared to other repressive chromatin regulators. They will develop a new framework and technology for linking epigenetic modulation to phenotype, and apply it to human stem-cell-derived cardiomyocytes subjected to environmental stressors. Photoswitchable small molecules (HDACi) and patterned light are used to imprint a dynamic epigenetic modification in time-space. Optically-gated high-pressure freezing will temporally-resolved precise registration of the immediate HDACi-mediated chromatin modulation, imaged by electron microscopy. Documenting the sequence of events, triggered by the epigenetic master-regulators of cell function, will have a broad impact for fundamental understanding of biological function, namely how does the genome re-arrangement link to phenotype under different stressors.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This project will study the problem of antibiotic-resistant bacterial infection associated with medical devices. The goal is to develop improved methodologies for quantitative assessment of this problem in the medical device context. This project will develop an inexpensive, reproducible and quantitative basis for comparing innovative therapeutic strategies to reduce the emergence of drug resistant bacteria. The knowledge gained in this work will help researchers in the area of regulatory science to improve predictive capability for the assessment of novel antibiotics. This work will also help to improve predictive capability for assessment of new therapies using existing antibiotics or combinations of existing antibiotics. Graduate and undergraduate students will have opportunities to develop their communication and leadership skills. The team will hold monthly interactive meetings at GWU or FDA.

The GWU-FDA collaborative team will develop an in vitro resistance evolution model based on microfluidics and on-chip optical sensing to assess how antibiotic resistance emerges in biofilm over 2 weeks on urinary catheters in response to clinically realistic pharmacokinetics/pharmacodynamics. This model will expose biofilm on catheters to 2-week concentration-time profiles similar to those experienced in vivo. The model aims to use lab-on-a-chip technology to increase the throughput of testing. This project will lead to new insight and improved mechanistic understanding of the special infection challenges that are posed by biofilms that are associated with indwelling and implanted medical devices. The PI will use recent developments in optical diagnostics and microfluidics technology.

Abstract Septic shock is invariably associated with systemic coagulation leading to thrombus formation. Sepsis-related organ dysfunction has been attributed to microvascular thrombosis. Mortality rate doubles in septic patients with disseminated intravascular coagulation (DIC). DIC is even considered as a sign that ?death is coming?. Previous studies have demonstrated the important roles of tissue factor (TF) in sepsis-associated DIC. However, the mechanism leading to TF release, which triggers systemic coagulation in sepsis, is unknown. Recent in vitro studies revealed that bacterial components (flagellin, the rod protein of the type III secretion system (T3SS), or LPS) induce programmed cell death (called pyroptosis) of macrophages through activation of inflammasome pathways. We show that intravenous injection of a T3SS rod protein E. coli, EprJ, induced depletion of peripheral monocytes and macrophages in tissues. Importantly, injection of EprJ or LPS, which elicit canonical and noncanonical inflammasome activation, respectively, induced systemic coagulation activation, as evident by prolonged prothrombin time (PT) due to increased consumption of coagulation factors, thrombocytopenia, increased plasma levels of thrombin-antithrombin complex (TAT), and reduced plasma fibrinogen levels. Thus, our findings made connections between the in vitro and in vivo observations and suggested monocyte/macrophage pyroptosis as a trigger of DIC in sepsis. The goal of this application is to delineate the underlying mechanisms by which inflammasome activation and pyroptosis trigger DIC in sepsis. Specific Aim 1 will establish inflammasome activation and pyroptosis as a common mechanism for DIC induced by bacterial infection. The working hypothesis is that bacteria and bacterial components from different strains elicit DIC through Inflammasome activation and pyroptosis. We will use a combination of various deficient mice to elucidate the role of inflammasome activation and pyroptosis in DIC elicited by Gram-negative bacteria. Specific Aim 2 is to identify the molecular mechanism of TF release from macrophages following inflammasome activation. We will also use the myeloid-specific TF knockout mice and a low TF mouse model to elucidate whether DIC elicited by the bacterial components depends on release of TF from macrophages. Specific Aim 3 will demonstrate the role of inflammasome activation in sepsis-associated coagulopathy. We will use the cecal ligation and puncture (CLP) sepsis model and bacterial infusion sepsis model to investigate the role of inflammasome activation and pyroptosis in coagulation. Completion of the proposed studies will demonstrate the molecular mechanism of systemic coagulation is sepsis. Such findings would have profound ramifications for the identification of new drug targets for DIC, the deadly complication of sepsis.

Sepsis is a life-threatening condition that affects more than 1 million patients a year in the United States. Growing evidence indicates that immunosuppression is a major driving force for mortality in sepsis. Macrophages play essential roles in immune response to pathogens. Previous clinical studies have shown that peripheral monocytes are depleted in septic patients through apoptosis. Recent in vitro studies revealed that bacterial components, flagellin, the rod protein of the type III secretion system (T3SS), or LPS induce pyroptosis of macrophages through activation of inflammasome pathways. In this proposal, we provide convincing evidence that both peripheral monocytes and tissue macrophages are depleted due to pyroptosis in mouse sepsis models including the cecal ligation and puncture (CLP) model. We show that intravenous injection of flagellin or the rod proteins induced depletion of peripheral monocytes and macrophages in tissues. We further demonstrate that depletion of these cells in mice impaired immune response and increased mortality rate by subsequent challenged with E. coli. Importantly, our data indicate that tissue factor released from pyroptotic monocytes and macrophages triggers disseminated intravascular coagulation (DIC). Thus, our findings identified monocyte/macrophage depletion as a novel mechanism of immunosuppression and DIC in sepsis. The goal of this application is to delineate the underlying mechanisms of monocyte/macrophage depletion and its contribution to immunosuppression in sepsis. Aim 1 is to delineate the mechanisms of pyroptotic monocyte and macrophage death during sepsis. The working hypothesis is that inflammasome activation and subsequent pyroptosis play a critical role in monocyte/macrophage depletion during sepsis. Mouse models deficient in caspase-1, caspase-11, caspase-1/11 double, or GSDMD (whole-body and macrophage-specific) will be used to elucidate the detailed mechanism of inflammasome activation and pyroptosis in monocyte/macrophage depletion. Aim 2 is to identify the mechanism by which rod protein and flagellin induce pyroptosis leading to monocyte/macrophage depletion. We recently identified an intracellular binding partner of EprJ, the acyl-CoA dehydrogenase family member type 9 (ACAD9) in macrophages. We will use different approaches to test the hypothesis that the ACAD9-dependent pathway contributes to macrophage depletion. Aim 3 is to identify the contribution of monocyte/macrophage depletion to immunosuppression during sepsis. We will use a combination of sepsis models to investigate the role of monocyte/macrophage depletion in immunosuppression during sepsis. Peripheral monocyte depletion in septic patients will also be investigated. Completion of the proposed studies will reveal a novel molecular mechanism of immunosuppression induced by Gram-negative bacteria. Such findings will significantly advance our understanding about the pathogenesis of sepsis and identify new drug targets for this deadly disease.

ABSTRACT FROM ORIGINAL PROPOSAL Sepsis is a life-threatening condition that affects more than 1 million patients a year in the United States. Growing evidence indicates that immunosuppression is a major driving force for mortality in sepsis. Macrophages play essential roles in immune response to pathogens. While depletion of T, B, and dendritic cells has been identified in septic patients, whether macrophages are depleted in sepsis remains to be determined. In this proposal, we provide convincing evidence that i.p. injection of E. coli or cecal ligation and puncture (CLP) treatment in mice induced peripheral monocyte depletion and macrophage death in tissues. Recent in vitro studies showed that inflammasome activation elicited by Gram-negative bacteria involves multiple mechanisms, including noncanonical inflammasome activation and pyroptosis by intracellular LPS and canonical inflammasome activation by flagellin and the rod protein of the type III secretion system (T3SS) (Fg. 1). We show that intravenous injection of flagellin or the rod proteins from several Gram-negative bacteria induced depletion of peripheral monocytes and macrophages in tissues. We further showed that mice pretreated with the E. coli T3SS rod protein EprJ or a sub-lethal dose of E. Coli impaired immune response of macrophages and increased mortality rate by subsequent challenge with E. coli. Based on our preliminary findings, we hypothesize that monocytes and macrophages are depleted during sepsis, which contributes to sepsis-induced immunosuppression. We have assembled a multidisciplinary team and established a variety of innovative models and assays to test this hypothesis. In Specific Aim 1, we will investigate the role of inflammasome activation and pyroptosis in monocyte/macrophage depletion during sepsis. We will use caspase 1, caspase 11, and caspase1/11 double knockout mice to determine whether inflammasome activation plays an important role in monocyte/macrophage depletion during sepsis. We will use Gsdmd whole- body and macrophage-specific knockout mice to determine whether pyroptosis plays a role in monocyte/macrophage depletion. In Specific Aim 2, we will elucidate the role of apoptosis in monocyte/macrophage depletion during sepsis. We will investigate whether caspase 8- or 3-dependent apoptosis contribute to monocyte/macrophage depletion during sepsis using caspase 8 and 3 deficient mice. In Specific Aim 3, we will identify the contribution of monocyte/macrophage depletion to immunosuppression during sepsis. We will use a combination of sepsis models combined with bacterial strains lacking T3SS rod proteins or/and flagellin to investigate the role of monocyte/macrophage depletion in immunosuppression during sepsis. Completion of the proposed studies will reveal a novel molecular mechanism of immunosuppression induced by Gram-negative bacteria. Such findings will significantly advance our understanding about the pathogenesis of sepsis and identify new drug targets for this deadly disease.