Jian Wang - US grants

Acutely, hypoxia increases pulmonary vasomotor tone, resulting in acute hypoxic pulmonary vasoconstriction (AHPV). Chronically, hypoxia causes pulmonary vascular remodeling and sustained increase in tone, resulting in chronic hypoxic pulmonary vasoconstriction (CHPV). The mechanisms of AHPV and CHPV remain unknown. Evidence suggests that calcium influx from extracellular fluid into pulmonary artery smooth muscle cells (PASMCs) plays a role. In many cells, depletion of Ca2+ stored in sarcoplasmic reticulum (SR)causes "capacitative Ca2+ entry (CCE)" through store-operated Ca2+ channels (SOCCs) composed of proteins homologous to "transient receptor potential" (TRP) proteins in Drosophila. Our preliminary data indicates that hypoxia increased resting [Ca2+]i and CCE in PASMCs and that TRPC1 and -6 gene expression in PASMCs was enhanced by hypoxia, perhaps due to activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). In this proposal, we test the hypotheses that in PASMCs acute hypoxia causes activation of CCE through sarcolemmal SOCCs composed of TRPC proteins, leading to increased [Ca2+]i and AHPV, while chronic hypoxia causes a sustained increase in CCE and resting [Ca2+]i in PASMCs due to HIF-1 dependent upregulation of TRPC protein expression, leading to CHPV. As initial tests of these hypotheses, we will determine: 1) if treatment of PASMCs with small interfering RNA (siRNA) specific for TRPC proteins blocks increases in [Ca2+]i and CCE caused by acute hypoxia;2) the effects of chronic hypoxia on TRPC expression, CCE and pulmonary vascular resistance;3) whether SOCC antagonists reverse hypoxia-induced changes in baseline [Ca2+]i, CCE and pulmonary vascular reactivity;4) if treatment with specific TRPC siRNA prevents changes in basal [Ca2+]i and CCE in PASMCs induced by prolonged hypoxia and 5) whether the transcription factor, HIF-1is responsible for hypoxia-induced upregulation of TRPC expression. We hope that our results will provide new mechanistic information and that elucidating the factors involved in this process will lead to improved methods of pharmacological prevention and treatment of this lethal complication of chronic lung disease.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this K01 award is to prepare the applicant for a career as an independent investigator in intracerebral hemorrhage (ICH) research. The applicant has shown a firm commitment to a biomedical research career and has acquired extensive training and expertise in molecular biology, histology, and animal models of ICH. He believes that enhancing his expertise in these areas and persuing additional training in in vitro experimentation and cellular neurobiology will improve his competitiveness. His mentor, Dr, Sylvain Dore, is an established neuroscientist in the areas of prostaglandin (PG) receptors, ischemic stroke, and aging, with expertise in primary cell cultures and cellular and molecular neurobiologic approaches. Dr. Solomon H. Snyderwill serve as an internal collaborator, while Drs. Eng H. Lo and Gary A. Rosenberg will serve as the consultants. They will provide him both intellectual and technical advice in their fields of expertise. The goal of this research is to understand the role of PGE2 receptors in the aging brain after ICH. We hypothesize that PGE2 EP1 and EPS receptors promote acute brain injury, whereas EP2 and EP4 receptors promote neuroprotection after ICH. Three specific aims will test our hypothesis. 1) Determine whether the PGE2 EP1 and EPS receptors increase brain injury in in vivo models of ICH, assess whether brain injury is accentuated in aged animals vs young ones, and assess the MMP-9 role; 2) Determine whether the PGE2 EP2 and EP4 receptors attenuate brain injury in in vivo models of ICH, assess the effect of aging on ICH outcomes, and assess the MMP-9 role; 3) Determine the effects of PGE2 EP1-4 receptor activation and inhibition on neuronal survival in in vitro models of heme-induced toxicity and assess the MMP-9 role. We will use the two mouse ICH models in our lab and our available EP receptor knockout C57BL/6 mice. Pharmacologic approaches (selective agonists and antagonists) for each of the receptors will be used to corroborate those findings from the knockouts. We believe that this work will provide a better understanding of specific PGE2 receptor functions in the aging brain after ICH. The data generated from this study will be strong enough to support the candidate's submission of an independent NIH R01 grant. RELEVANCE (See instructions): The work proposed will provide a better understanding of specific prostaglandin E2 receptor functions in the aging brain after intracerebral hemorrhage. Insight into these receptors may contribute to the basis for therapeutic intervention for intracerebral hemorrhage in the elderly. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chronic hypoxia (CH)-induced sustained increases in vascular tone and pulmonary vascular remodeling play key roles in the pathogenesis of chronic hypoxic pulmonary hypertension (CHPH). Despite progresses have been made on exploring the role of Ca2+ in these processes, the underlying molecular mechanisms, however, remain largely unknown. Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor-2 (TGF-2) superfamily, are known as critical regulators in mammalian development, cell proliferation, differentiation and apoptosis. Recently, the identification of germline mutation of BMP receptor II (BMPRII) in familial pulmonary hypertension and other associated group of evidence indicate the implication of abnormal BMP signaling in the pathogenesis of pulmonary hypertension. In particular, ug-regulation of bone morphogenetic protein 4 (BMP4) by CH was suggested to be an important factor that influences the development of CHPH. We previously demonstrated that CH elevated basal intracellular [Ca2+] ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) due in large part to enhanced store-operated calcium entry (SOCE) through store-operated Ca2+ channels (SOCCs) likely composed of canonical transient receptor potential proteins (TRPCs). In our recent studies, we obtained data showing a role of BMP4 in regulation of TRPCs expression and Ca2+ influx. These data include: 1) BMP4 treatment increased TRPC1 and TRPC6 expression, SOCE and basal [Ca2+]i in PASMCs;2) Exposure to CH increased mRNA and protein expression of TRPC1 and TRPC6, SOCE and basal [Ca2+]i in PASMCs, and these CH-induced increases were attenuated by knockdown of BMP4 expression via specific BMP4 siRNA, or BMP4 depletion using its antagonist noggin;2) CH enhanced both mRNA and protein expressions of BMP4 in mouse lung;3) Overexpression of HIF-1a increased BMP4 expression in PASMCs, and the CH-induced increases of BMP4 expression were impaired in HIF-1a partially deficient mice. These results suggest that BMP4 participate in the regulation of Ca2+ homeostasis in PASMCs during CH via modulation of TRPC channels, acting either in downstream of HIF-1a or in concert with HIF-1a dependent up-regulation of TRPCs. On the basis of the above findings and some other data in our studies, we hypothesize that the increased [Ca2+]i in PASMCs caused by CH is due to or partially due to HIF-1 dependent upregulation of BMP4, which leads to increases in TRPCs expression, SOCE and basal [Ca2+]i in distal PASMCs, thereby contributing to CHPH. To test this hypothesis, we will perform experiments in lung, PA and/or PASMCs using the combined techniques of microfluorescence measurements and molecular biology to accomplish the following specific aims: 1) Determine the roles of HIF-1 and BMP4 in up-regulation of TRPCs expression during CH;2) Determine BMP4 receptors and antagonist(s) that are responsible for hypoxic increases of TRPCs expression;3) Determine the signaling pathway through which BMP4 regulates TRPCs expression in PASMCs;4) Determine which TRPC contributes to the increases of SOCE and basal [Ca2+]i in response to CH. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension (PH) is a progressive devastating disease characterized by high blood pressure in the lungs;its mechanisms remain poorly understood. Hypoxia, an important trigger of PH, has been found to enhance calcium signaling in pulmonary artery smooth muscle cells, causing cell proliferation and constriction. Our study focuses on investigation of whether and how BMP4 regulates this process, which, if successful, will lead to improved methods of pharmacological prevention and treatment of this lethal complication of chronic lung diseases.

DESCRIPTION (provided by applicant): Neuroprotective effect of flavanol (-)-epicatechin after intracerebral hemorrhage Dietary polyphenols such as flavanols have been reported to help prevent diseases of the cardiovascular and central nervous systems. Cocoa and green tea are rich in flavanols, including (-)-epicatechin. A recent study from colleagues in our stroke group demonstrated that the flavanol (-)-epicatechin given orally reduces ischemic stroke damage through activation of Nrf2, a transcription factor that regulates the expression of endogenous antioxidant enzymes in the brain. We have demonstrated that mice lacking Nrf2 are more susceptible than wild-type control mice to brain damage from intracerebral hemorrhage (ICH). Additionally we showed that the exacerbation of brain injury in Nrf2 knockout mice was associated with increases in production of reactive oxygen species. The overall objective of this R01 is to address whether the flavanol (-)-epicatechin can be used as neuroprotective therapy in ICH, and if so, to generate preclinical efficacy data for its use and elucidate the underlying mechanisms. Our working hypothesis is that (-)-epicatechin reduces ICH injury through the Nrf2 pathway. We have designed three specific aims that utilize the collagenase-induced and autologous blood models of ICH. This research will be carried out in young and aged mice to enhance the clinical relevance. Aim 1 will determine whether daily (-)-epicatechin treatment after ICH improves outcomes in young male mice. Aim 2 will determine whether post-treatment with the optimal dose of (-)-epicatechin is effective in young female and aged mice. Aim 3 will determine whether the Nrf2 pathway contributes to the neuroprotective effect of (-)- epicatechin in ICH models and in in vitro models of hemoglobin-induced toxicity. Through pharmacologic, genetic, imaging, histologic, molecular, and cellular biologic approaches, we will provide novel information about the efficacy of (-)-epicatechin in the two mouse ICH models and about the underlying mechanisms. Such information is required to plan more detailed preclinical trials and could influence clinical practices regarding flavanol use. Ultimately, the data may help the general public and healthcare providers make informed decisions on whether (-)-epicatechin could be accepted as an adjunct treatment in patients with ICH.

DESCRIPTION (provided by applicant): PGE2 EP1 and EP3 receptors as therapeutic targets in intracerebral hemorrhage Intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity; unfortunately, this condition has received far less research attention than has ischemic stroke. Inflammatory mechanisms, including those mediated by certain prostaglandins (PGs), have been suggested to contribute to the progression of ICH injury. In particular, PGE2, which is predominant in the brain, is produced and accumulates in the perihematomal region. PGE2 acts through four G-protein-coupled receptor subtypes known as EP1- EP4. Each of these receptors has distinct signaling cascades in animal models of cerebral ischemia. Genetic deletion or selective inhibition of the EP1 and EP3 receptors has been shown to reduce ischemic brain injury both in vivo and in vitro. The overall objective of this R01 is to investigate whether inhibition of PGE2 EP1 and EP3 receptors can be used as neuroprotective therapy in ICH, and if so, to generate preclinical efficacy data and elucidate the underlying mechanisms. We have been investigating the specific roles of PGE2 receptors in ICH injury and have preliminary data showing that deletion or inhibition of the EP1 or EP3 receptor reduces ICH injury and improves functional outcomes. Our data also indicate that Src kinase mediates EP1 toxicity and Rho kinase (ROCK) mediates EP3 toxicity after ICH. Consequently, we will test the hypothesis that PGE2 EP1 receptor blockade reduces ICH injury through the Src kinase signaling pathway, whereas EP3 receptor blockade reduces ICH injury through the ROCK signaling pathway. We have designed three specific aims that utilize the autologous blood ICH model and collagenase-induced ICH model. Considering that an increase in PGE2 production contributes to age-related dysfunction of the inflammatory responses and that aging and sex differences affect ICH outcomes, this research will be carried out in young male and female and aged male mice to enhance the clinical relevance. Aim 1 will determine whether inhibition of EP1 or EP3 receptors improves ICH outcomes; Aim 2 will determine whether inhibition of Src kinase signaling contributes to the neuroprotection afforded by EP1 receptor blockade after ICH and whether EP1 receptor inhibition decreases thrombin-induced brain damage; and Aim 3 will determine whether inhibition of ROCK signaling contributes to the neuroprotection afforded by EP3 receptor blockade after ICH and whether EP3 receptor inhibition decreases thrombin-induced brain damage. Through pharmacologic, genetic, imaging, histologic, molecular, and cellular biologic approaches, this work will provide novel information about the efficacy of selective PGE2 EP1 and EP3 receptor inhibition to protect against ICH-induced brain injury and the mechanisms by which such protection occurs. This proof-of-concept information is required to plan more detailed preclinical assessment of selective EP1 and EP3 receptor antagonists in ICH.

PROJECT SUMMARY: At least one-third of stroke survivors suffer from post-stroke depression (PSD), which has been shown to negatively affect prognosis. Although PSD after ischemic stroke has been studied, very little is known about the frequency and severity of depression or depressed mood associated with intracerebral hemorrhage (ICH). However, patients who experience ICH do develop PSD. Identifying new drug targets for PSD specific for ICH patients is a pressing need because antidepressants, particularly selective serotonin reuptake inhibitors, are associated with an elevated risk of bleeding, increased stroke severity, and increased mortality from stroke or ICH. In an effort to limit ICH injury and improve functional recovery, we have investigated PSD in preclinical ICH models and shown for the first time that the mouse cortical ICH model produces depression-like behavior. Clinically, however, ICH occurs most commonly in the basal ganglia, an area that contributes to the development of PSD. Indeed, our pilot data showed that striatal ICH does lead to depression-like behavior in mice. The pathogenesis of PSD is complex, but a heightened inflammatory response and increased reactive oxygen species (ROS) production might be two key biochemical factors. The transcription factor Nrf2 is a master regulator of ROS and inflammation. Enhancing this endogenous system might provide a mechanism to reduce PSD after ICH. We have reported that Nrf2 activity is neuroprotective after ICH. We have also shown that the exacerbation of brain injury in Nrf2 knockout mice is associated with increased inflammation and ROS production and that the brain-permeable, Nrf2 inducer (?)-epicatechin offers neuroprotection against ICH. Brain-derived neurotrophic factor (BDNF) is one of the Nrf2 target genes, and Nrf2/BDNF signaling was shown to be involved in the antidepressant-like effect produced by agmatine and fluoxetine. Because the neurobiologic mechanisms of PSD may differ from those of other depression subtypes, it is unknown whether deletion/activation of Nrf2 exacerbates/mitigates PSD after striatal ICH. The overall objective of this R21 is to investigate whether the striatal ICH model can be used to investigate PSD after ICH, and if so, to determine the role of the Nrf2/BDNF pathway in the pathogenesis of PSD. In two specific aims, we will test the hypothesis that dysregulation of the Nrf2/BDNF pathway contributes to the development of PSD after ICH. Aim 1 will determine whether striatal ICH produces PSD in young mice of both sexes, and Aim 2 will determine whether dysregulation of the Nrf2 pathway contributes to PSD after ICH. Successful validation of the striatal ICH model as an appropriate approach to investigate PSD will provide the rationale for expanded preclinical studies on PSD. This project is highly clinically relevant and represents the first preclinical evaluation of PSD in a striatal ICH model. The results will provide insight into the molecular mechanisms of PSD after ICH and will identify and validate new therapeutic targets for ICH-induced PSD, an under-investigated clinical problem identified by the American Heart Association (Stroke 2017; 48: e30-e43).

PROJECT SUMMARY: Microglia/macrophage polarization after intracerebral hemorrhage Spontaneous intracerebral hemorrhage (ICH) causes high mortality and morbidity, but it is understudied compared to ischemic stroke, and effective treatment is lacking. Hematoma volume and expansion are independently associated with poor patient outcomes; therefore, rapid removal of toxic blood could limit ICH- induced brain injury. After ICH, microglia and macrophages (MM?) shift activity states to remove toxic blood and may protect the brain. However, over-activated MM? cause secondary brain damage by releasing cytotoxic substances. These opposing effects may result from distinct MM? subsets, which are categorized into classically activated proinflammatory (M1) and alternatively activated anti-inflammatory (M2) cells. Alternatively activated M2 MM? exhibit increased phagocytosis of apoptotic cells, which could involve activation of the scavenger receptor CD36 and inhibition of its negative regulator toll-like receptor (TLR)4. Additionally, interleukin-10 (IL- 10), an anti-inflammatory cytokine, represses inflammation, polarizes macrophages to an M2c subtype, and enhances phagocytosis. High levels of IL-10 in blood or brain tissue have been reported in ICH patients. However, MM? polarization and the exact role of IL-10 signaling in M2 polarization after ICH are unknown. The long-term goal of our research is to limit ICH injury and improve functional outcomes. The overall objective of this R01 is to investigate whether modulation of MM? phenotype and function by IL-10 reduces ICH injury and improves histologic and functional outcomes. Our preliminary studies showed that IL-10 expression increases in brain slice cultures exposed to hemoglobin and in an in vivo model of ICH; that IL-10 increases microglial phagocytosis and CD36 expression in brain slice cultures; that IL-10-deficient mice have impaired hematoma resolution and altered CD36 and TLR4 expression compared with that in C57BL/6 wild-type mice; and that exogenous IL-10 successfully reduces hematoma volume. These findings prompt the hypothesis that polarizing MM? to M2 phenotype by IL-10 reduces ICH injury and improves histologic and functional recovery after ICH. In three specific aims, we will determine whether M2 microglial polarization by IL-10 is responsible for phagocytosis in ex vivo brain slice cultures exposed to hemoglobin or aged red blood cells (Aim 1); whether M2 MM? polarization by IL-10 improves the histologic and functional outcomes after ICH in vivo (Aim 2); and whether IL-10-induced MM? M2 polarization requires activation of CD36 and inhibition of TLR4. The information gained from this study will provide us with novel insight into the MM? polarization after ICH and the cellular and molecular mechanisms by which IL-10 signaling?induced MM? M2 polarization reduces ICH injury. Based on multidisciplinary approaches, our findings will potentially lead to a new therapeutic strategy not only for ICH but also for other brain disorders. This novel proof-of-concept work to study modulation of innate inflammation is a critical priority identified by the recent NINDS-SPRG.

ABSTRACT Traumatic brain injury (TBI) is an important public health problem. Currently, the ability to objectively assess TBI is a critical research gap. CT and conventional structural MRI have proven to be highly effective in identifying macroscopic lesions. However, these standard imaging techniques have clear limitations in assessing important microscopic lesions and neurologic pathology. The clinical diagnosis of TBI via imaging, particularly mild TBI, remains controversial, because the brain often appears quite normal on conventional CT and MRI. Therefore, new sensitive surrogate biomarkers for TBI are greatly needed in routine clinical practice. Amide proton transfer (APT) imaging is an important molecular MRI technique that can generate contrast based on tissue pH or concentrations of endogenous mobile proteins and peptides. In our preliminary study, we have applied the APTw-MRI approach to a rat TBI model, induced by controlled cortical impact (CCI). Our preliminary results have demonstrated unique APTw-MRI signal characteristics at different time points after injury that are associated with ischemia (at a few hours) and neuroinflammation (at 2-3 days). Notably, APT imaging revealed an acidosis-based ischemic penumbra around the impacted area at a few hours post-injury. These initial results are very promising. However, further development and radiographic-histopathologic validation with different TBI models and at other research sites is crucial for translating this innovative technology and these important results to the clinic. The overall goal of this application is to demonstrate the feasibility, potential, and reproducibility of protein-based APT-MRI signals as functional markers for TBI using animal models of mild, moderate, or severe TBI. We hypothesize that molecular imaging using APT-MRI can sensitively and non-invasively visualize ischemic damage, inflammatory responses, and several other key pathological processes in TBI, thus improving the capability of MRI to objectively assess TBI. Our three specific aims are: (i) to assess APT-MRI spatio-temporal evolution characteristics of TBI and the underlying pathological mechanisms in rat CCI models; (ii) to assess whether APT-MRI predicts therapeutic outcomes in rat CCI models; and (iii) to validate the sensitivity and reliability of APT-MRI in assessing TBI at external sites. Molecular imaging of TBI using APT-MRI opens up a new research area of APT imaging that could address many unmet clinical needs. If our aims in this preclinical study are achieved, the results will provide the solid foundation required to translate this important MRI technology to clinical studies in patients with TBI.

Inhibition of ferroptosis after intracerebral hemorrhage Spontaneous intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity. Because large amounts of blood are released into the extracellular space during ICH, the metabolism of hemoglobin/free heme is very important for brain recovery. Heme is degraded by heme oxygenase into iron, biliverdin, and carbon monoxide. Importantly, iron accumulation within the brain contributes to secondary brain injury after ICH. Indeed, iron toxicity contributes to collagenase-induced hemorrhagic brain injury in mice, and reducing iron accumulation with iron chelators can improve neuronal survival. Recently, ferroptosis, an iron- dependent form of non-apoptotic cell death, was identified in cancer cells and in organotypic hippocampal slice cultures after glutamate exposure. It is triggered by small molecules or by conditions that inhibit glutathione biosynthesis or glutathione peroxidase 4 (GPX4) activity. This regulated cell death is characterized by extensive iron-dependent lipid peroxidation, which can be suppressed by lipophilic antioxidant ferrostatin-1 or liproxstatin- 1. Ferroptotic cell death also has been shown to occur in brain cells. To date, it has been reported in mouse models of Parkinson disease and ICH. Whether inhibition of ferroptosis by ferrostain-1 or liproxstain-1 reduces ICH injury and improves functional outcomes in aged animals remains unknown. Therefore, this research is intended to characterize ferroptotic cell death in the context of ICH in aged animals. Our long-term goal is to limit ICH injury and improve functional outcomes. The scientific objective of this proposal is to test the hypothesis that inhibition of ferroptosis by ferrostain-1 or liproxstain-1 protects ICH brain and improves functional outcomes. We will further determine whether augmentation of GPX4 activity inhibits ferroptosis and provides cerebroprotection after ICH. In pilot studies, ferroptotoic cell death, characterized by shrunken mitochondria, was detected by transmission electron microscopy in the mouse ICH brain. Additionally, mice treated with ferrostatin-1 after ICH had better neurologic outcomes than mice treated with vehicle. A second, independent, ferroptosis inhibitor, liproxstatin-1, also exhibited marked protection against ICH injury. Together, these novel observations strongly support the premise that inhibition of ferroptosis might reduce ICH injury in aged animals. The first specific aim will determine whether suppression of iron-dependent lipid peroxidation improves histologic and functional outcomes after ICH in aged mice of both sexes. The second specific aim will determine whether overexpression of GPX4 is cerebroprotective after ICH. This study will provide novel evidence that ferroptotic cell death, in addition to other regulated cell death pathways, is prominent in the brains of aged animals with ICH. It will also provide insight into the molecular mechanism by which increased GPX4 activity prevents post-ICH ferroptosis. Work on the proposed project could render new drugs/treatments for patients with ICH. Successful validation of lipid peroxidation inhibitors in the ICH models of aged animals will provide the rationale and proof-of-concept for future preclinical and clinical trials.