John Y. Shyy - US grants

DESCRIPTION: (Adapted from investigator's abstract) The objectives of the proposed research entitled "Molecular Basis of the Endothelial Shear Stress Receptor" are to elucidate the mechanisms of mechano-biochemical transduction through the study of receptor tyrosine kinases (TRKs) serving as shear stress receptors and the signal transduction through which the downstream genes are activated. We have been able to demonstrate in previous studies that the phorbol ester TPA responsive element (TRE) is a shear stress inducible cis-element. Our preliminary results illustrate further that p21ras is upstream to such TRE-mediated gene expression. In addition, the epidermal growth factor receptor (EGFR) is phosphorylated in response to shear stress. By using EGFR as a model RTK in conjunction with in vitro flow channel experiments, the proposed studies are designed to provide linkage between two membrane-associated events and to correlate the structural features of the extracellular domains of TRKs with the shear stress-induced cellular responses. In Specific Aim 1, we will investigate the recruitment of the src homology domain 2 (SH2)-containing molecules, including growth factor receptor binding protein-2 (Grb2), Shc, Son of sevenless (Sos), and RasGTPase activating protein (RasGAP) to the phosphotyrosines of the shear stress-activated RTKs. While the assembly of RTKs-Shc-Grb2-Sos quaternary complex and RTKs-Grb-2os ternary complex activates the Ras signaling pathway, the formation of RTKs-Ras-GAP binary complex antagonizes the activation of Ras. The resulting transient cellular responses will be examined by assessing the activities of p21ras, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and the luciferase reporter activities driven by TRE. In Specific Aim 2, these cellular responses will be studies in cell lines expressing EGFR mutants and fusion receptors. The extracellular domains of EGFR in these molecules are either mutated or replaced with those of other RTKs to investigate their function as shear stress sensors. The effects of shear stress will be compared with those of ligand binding with respect to the tyrosine phosphorylation and receptor dimerization. In Specific Aim 3, we will examine whether the deduced molecular mechanisms are also functional in the vascular endothelial cells which are the cell type exposed to pathophysiological flow conditions in the body. The proposed research has considerable significance in elucidating both the fundamental mechano-biochemical transduction processes and the endothelial biology in atherogenesis.

Mechanical loading in hypertension and other vascular diseases can lead to vascular remodeling which involves proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The major hemodynamic force borne by VSMCs in physiological and pathophysiological conditions is the circumferential stress which results in mechanism strain. Three specific aims are proposed to test our hypothesis that mechanical strain causes augmentation of both proliferation and apoptosis through mechanotransduction mechanisms in which integrins function as mechanotransducers to activate the mitogen-activated protein kinases (MAPKs) pathways. In Specific Aim 1, we will determine the roles of the mechanical strain-activated MAPKs signaling pathways in modulating the VSMC proliferation and apoptosis. We propose that activation of the ERK (i.e., extracellular signal-regulated kinases) pathway is critical for mitotic events, whereas augmentation of the JNK (i.e., c-Jun N-terminal kinases) pathway is important for apoptotic responses. We will investigate whether the various negative mutants of signaling molecules in the ERK pathway can block the strain-induced VSMC proliferation, and whether mutants in the JNK pathway can abolish VSMC apoptosis. In Specific Aim 2, we will use alphavbeta3 integrin as a model to study the functional roles of alphav and beta3 units in mechanical strain activation of ERK and JNK. Using monoclonal antibodies specific for integrins in their activation state, we will further test whether alphavbeta3 can be directly activated by mechanical strain. In Specific Aim 3, we will delineate the signaling pathways linking integrins to MAPKs in response to mechanical strain. We propose that the action of mechanical strain on integrins/ECM complexes (e.g., alphavbeta3/vitronectin) triggers the association of integrins with Shc and Grb2. This event in conjunction with the increase in the kinase activities of FAK and c-Src, results in the activation of the MAPKs pathways. In all three Specific Aims, in vitro experiments will be conducted using a newly designed stretch device to apply uniform equi- biaxial strain to human aortic smooth muscle cells cultured on silicon elastic membrane. This interdisciplinary research will generate new insights into the molecular mechanism by which mechanical strain leads to accelerated mitosis and apoptosis of VSMCs and thus enhance our understanding of the pathophysiological basis of vascular remodeling resulting from alterations in mechanical strain.

DESCRIPTION (Adapted from Applicant's Abstract): Percutaneous transluminal coronary angioplasty (PTCA) can cause the denervation of epithelium and the injury to medial vascular smooth muscle cells (VSMCs). In response to the imposed injury, VSMCs migrate from the media to the intima, which is an important pathogenic event of restenosis. Preliminary studies with use of flow channel show that the Cbl, which is a proto-oncogene product (Sasitas B-lineage lymphoma), and is the cellular homology of the alcoprotein b-Cbl, scaffolding molecule and Rho and Rac GTPases play clinical roles in enhancing VSMC migration in response to shear stress. Thus, we hypothesize that integrins in VSMCs serve as mechanotransducers by dynamically interacting with specific extracellular matrices (ECMs). As a result, Cbl is tyrosine-phosphorylated to form a complex with PI3K and Vav2, which, in turn, activates the Rho-ROCK and Rac-gelsolin pathways. The coordination of these two pathways enhances the migration of VSMCs. Four Specific Aims are proposed to test our hypotheses. Specific Aim 1 is to elucidate the roles of integrin in shear stress enhancement of VSMC migration. Specifically, we will study the mechanically sensitive integrins and VSMCs, and the test the hypothesis that a specific and dynamic interaction between these integrins and their cognate ECMs is important in augmenting VSMC migration. Specific Aim 2 is to delineate the molecular mechanisms by which Cbl activates Rho and Rac to enhance VSMC migration. Specifically, we will examine whether the Cbl functions as a scaffolding molecule to dock PI3K and Vav2 in activating Rho and Rac, and investigate whether shear stress activation of Cbl-PI3K-Vav2 depends on integrin and ECM interaction. Specific Aim 3 is to determine the roles of the Rho-ROCK and Rac-gelsolin signaling pathways in shear stress enhancement of VSMC migration. Specifically, we will explore the role of Rho in activating ROCK, and the role of Rac in activating gelsolin to enhance VSMC migration. Specific Aim 4 is to investigate the roles of Cbl, Rho, and Rac in VSMC migration in rat carotid arteries after balloon angioplasty. Specifically, we will examine the activation of Cbl, Rho and Rac in injured vessels and test the efficacy of using negative and active mutants of these molecules to inhibit or enhance VSMC migration in vivo. With strong interaction between Bioengineering, Surgery and Biomedical Sciences, as well as in vitro and in vivo approaches this proposal will generate new information relevant to mechanotransduction in VSMCs and pathophysiology of restenosis.

DESCRIPTION (provided by +applicant): The focal nature of atherosclerosis at the bifurcations and curved regions of arterial tree is correlated with the local flow patterns imposed on vascular endothelial cells (ECs). We hypothesize that disturbed flow provides a hemodynamic milieu to activate sterol regulatory element binding proteins (SREBPs) in ECs and modulate the expression and function of genes involved in lipid metabolism and trafficking. Synergistic with hyperlipidemia, disturbed flow causes the accumulation of LDL and its metabolites in the subendothelial space, vascular smooth muscle cells (VSMCs), and macrophages. In contrast, steady flow protects the endothelium by preventing SREBP activation, thus maintaining lipid homeostasis in straight parts of the arterial tree. Four specific aims are proposed to test our hypotheses. Specific Aim 1 is to apply flows with variations in magnitude of shear stress, temporal and spatial shear stress gradients, and amplitude and frequency of pulsatility to ECs cultured in flow channels. We will profile SREBP activation and transcriptional regulation of genes involved in lipid metabolism and trafficking in response to the various flow patterns. Specific Aim 2 is to investigate the synergistic effect of flow activation of SREBPs and high levels of LDL in the subendothelial accumulation of lipids. Flow conditions that can cause sustained SREBP activation will be applied to EC-VSMC or EC-macrophage co-culture systems under high levels of LDL The lipid accumulation in the subendothelial space, VSMCs, and macrophages will be investigated accordingly. Specific Aim 3 is to compare the topographic distribution of flow-activated SREBPs and atherosclerofic lesions in the arterial tree in rive. We will create transgenic mice possessing an EC-specific SRE-LacZ reporter system. The topographic expression of LacZ regulated by arterial flow will be compared with the distribution of atherosclerotic lesions in apoE -/- mice. Specific Aim 4 is to explore the role of SREBPs in focal pattern of atherosclerosis. Transgenic lines with either EC-specific knockout of SREBP cleavage-activating protein (SCAP) or overexpression of the active form of SREBP2 [SREBP2(N)] will be created and crossed with apoE mice. We will examine whether atherosclerotic lesions in arterial branches can be attenuated in SCAP knockout animals (loss-of-function) and whether mice overexpressing SREBP2(N) show an enhancement of atherogenesis throughout the arterial tree (gain-of-function). By combining bioengineering, vascular biology, and transgenic approaches, this program will generate new insights into the molecular and mechanical bases of local flow patterns in atherogenesis.

DESCRIPTION (provided by applicant): Acting on multiple tissue types, adiponectin (Adipo) exerts insulin-sensitizing and cardiovascular protective effects. The physiological and/or clinical implications are that the plasma level of Adipo is negatively related to adiposity, insulin resistance, and cardiovascular disease, but weight reduction or increased exercise significantly elevates Adipo levels in diabetic or obese individuals. In vascular endothelium, Adipo binding to the Adipo receptors elicits AMP-activated protein kinase (AMPK) to phosphorylate/activate endothelial nitric oxide synthase (eNOS), which enhances NO bioavailability. Compared with the wild-type human Adipo, mutation of 2 amino acids resulted in high expression by both bacterial- and mammalian cell-based systems and high solubility in saline. Indeed, this newly created recombinant protein, designated Adipo3962, could be produced in large quantity, with in vitro and in vivo bioavailability comparable to that of the wild-type Adipo. Using Adipo3962, we found that Ca2????dependent protein kinase kinase (CaMKK) is the upstream kinase phosphorylating AMPK responding to Adipo. In addition, we developed a prototype of a fluorescence resonance energy transfer (FRET)-based AMPK activity reporter (AMPKAR) to measure AMPK activity. From these preliminary results, we propose this R21 pilot study, with emphasis on two specific aims: Specific Aim 1 will establish a cell-based system to assess the AMPK activation. We will create a vascular endothelial cell (EC) line stably expressing AMPKAR. Activation of the CaMKK-AMPK axis in EC-AMPKAR cells will be assessed by the FRET response. Specific Aim 2 will investigate the Adipo-activated CaMKK-AMPK and the associated beneficial effects in mouse vessels. We will administer clinical doses of Adipo3962 to Adipo-/-, CaMKK2-/-, and AMPK11-/- mice. The AMPK activity in the mouse aorta and the consequent vascular tone (i.e., blood pressure, NO bioavailability, aortic ring relaxation) will be investigated. Thus, the goal of this R21 proposal is to improve the AMPKAR prototype to accurately measure AMPK activity in vitro and in vivo and to characterize the role of CaMKK-AMPK signaling in endothelial functions benefiting from Adipo. PUBLIC HEALTH RELEVANCE: Cardiovascular impairment associated with metabolic diseases has reached epidemic proportions in recent years. Because of the important role of adiponectin and AMP-activated kinase (AMPK) in regulating vascular function and metabolism, we propose to establish an AMPK biosensor that can accurately measure AMPK activity responding to various pharmacological agents, including a mass-produced recombinant adiponectin. The proposal will increase our understanding of the mechanisms by which adiponectin activates AMPK and the utility of the recombinant adiponectin in cardiovascular protection.

DESCRIPTION (provided by applicant): The prevalent distribution of atherosclerotic lesions in the curvature and branches of arterial trees demonstrates the critical role of flow patterns in predisposing the arterial wall to atherosclerosis. Steady flows with a high magnitude of shear stress, the tangential component of hemodynamic forces acting on the endothelium, constitute the waveform of atheroprotective flow. In contrast, flow patterns with a low magnitude of shear stress, oscillation, and reverse flow, are atheroprone. Flow channels have been used as in vitro model systems to demonstrate that shear stress regulates the expression of genes involved in vascular functions. In investigating the mechanotransduction mechanisms, we recently found that AMP-activated protein kinase (AMPK) is modulated by shear stress in vascular endothelial cells (ECs). Moreover, Kr¿ppel-like factor 2 (KLF2), a zinc finger transcription factor integrating multiple endothelial functions, is regulated via AMPK in response to shear stress. We thus hypothesize that atheroprotective flow modulates the activity of AMPKK [i.e., calmodulin-dependent protein kinase kinase (CaMKK) and LKB1], which in turn activates AMPK. In addition to rapidly activating eNOS, AMPK augments the transcriptional activation of the klf2 gene to benefit the EC-dependent vascular functions. The activated AMPK-KLF2 would also contribute to the atheroprotective effect of flow. To test our hypothesis, three Specific Aims are proposed. Specific Aim 1 will dissect the critical shear stress parameters associated with the atheroprotective flow that activates the AMPKK-AMPK cascade in ECs. Specific Aim 2 will elucidate the molecular mechanism by which AMPK regulates the transcriptional activation of KLF2, which in turn modulates KLF2-targeted genes. Specific Aim 3 will investigate the role of flow-activated AMPK and KLF2 in vascular tone and atheroprotection. Specifically, we will assess the EC-dependent vessel dilation and NO bioavailability in AMPK knockout (ampk-/-) mice. Further, ampk-/- mice will be crossbred with apoE-/- mice. Atherosclerotic lesions are expected to be enhanced in these double knockout mice, as compared with the apoE-/- littermates. Aiming at studying post-translational and transcriptional regulation by AMPK in ECs exposed to shear stress, we hope this research will establish a framework to further understand the mechano and molecular basis of EC biology regulated by flow. PUBLIC HEALTH RELEVANCE: Atherosclerosis shows a focal pattern of distribution, which is mainly contributed by the distinct flow patterns depending on the locations in the arterial tree. The studies in this proposal address questions regarding the mechano and molecular basis of atheroprotective versus atheroprone flows in modulating the vascular endothelial biology. The proposal will significantly increase our understanding of mechanisms by which blood flow patterns interplay with hyperlipidemia in atherogenesis, which are likely to contribute to novel therapies for its prevention and/or treatment.

DESCRIPTION (provided by applicant): Arterial stiffening, manifested by thickening and reduced elasticity of conduit arteries, is an independent risk factor for stroke and heart disease. Although the etiology of arterial stiffening correlates well with aging, the underlying cellular and molecular basis remains unclear. In vivo, hemodynamic forces dynamically act on the vascular wall, which greatly affect arterial function and mechanical properties. Arterial stiffening may involves aberrant endothelial response to shear stress resulted from these hemodynamic forces. We recently found that SIRT1, an anti-aging regulator is modulated by shear stress in vascular endothelial cells (ECs). Moreover, calmodulin-dependent protein kinase (CaMKK) appears to be a shear stress-sensitive kinase that regulates SIRT1. We thus hypothesize that shear stress upregulates SIRT1 in the endothelium of conduit arteries, which ameliorates pathophysiological remodeling leading to arterial stiffening. At the upstream, CaMKKb is activated in response to the physiologically relevant shear stress. As a consequence of the activated CaMKKb-SIRT1 pathway, the endothelial nitric oxide synthase (eNOS)-derived NO bioavailability and PGC-1a-regulated ROS scavenger expression are augmented. Thus, the advantageous effects of shear stress-augmented SIRT1 include decreased oxidative stress and remodeling of extracellular matrices. To test our hypothesis, three Specific Aims are proposed. Specific Aim 1 will examine the hemodynamic factors critical for the induction of SIRT1 in cultured ECs. Molecular signaling experiments will then be conducted to study the mechanism by which CaMKKb regulates SIRT1 in ECs responding to the defined shear stress. Specific Aim 2 will elucidate the cellular and molecular mechanisms by which shear stress-induced SIRT1 exerts anti- stiffening effects in an EC/vascular smooth muscle cell co-culture system. We will decipher the synergistic effect of SIRT1 and AMP-activated protein kinase (AMPK) in eNOS-derived NO bioavailability and PGC-1a-regulated reactive oxygen species (ROS) scavengers. Specific Aim 3 will investigate the role of shear stress-activated SIRT1 in arterial stiffening in mouse models. Specifically, we will compare the spatiotemporal changes of arterial stiffening in EC-sirt1-/- knockout, Tg-EC-sirt1, and CaMKKb-/- mice. The arterial stiffening-associated changes in hemodynamic factors, aortic wall remodeling, and gene expression profiles will be measured and correlated. Results from these proposed studies, if as anticipated, will help to understand the mechano and molecular basis of arterial stiffening. PUBLIC HEALTH RELEVANCE: Arterial stiffening, associated with the aging process, is an independent risk factor for many cardiovascular diseases. SIRT1 is a master regulator involved in many anti-stress responses and we recently found that hemodynamic force can activate SIRT1 in vascular cells. This proposed study will investigate the mechano and molecular basis of flow induction of SIRT1, which would be anti-arterial stiffening. The proposal will significantly increase our understanding of mechanisms by which blood flow interplays with aging process in arterial stiffening. Results from the proposed experiments are likely to contribute to novel therapies for its prevention and/or treatment.

DESCRIPTION (provided by applicant): MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in regulating mRNA stability and translational repression. There is increasing evidence that miRs can modulate gene expression in the cardiovascular system. Endothelial cells (ECs) lining the vascular lumen are sensitive to mechanical factors such as fluid shear stress. During the past two decades, this research team has worked on the mechanisms of mechanotransduction in ECs and the consequent gene expression. The results from them and others indicate that steady and pulsatile shear stresses (PS) with a net forward direction are anti-atherogenic by inducing genes involved in anti-proliferation and anti-inflammation. In contrast, oscillatory shear stress (OS) without a significant forward direction is pro-atherogenic by activating pro-proiferative and pro-inflammatory genes. Based on new evidence in the literature and our recent findings that miRs play an important role in regulating EC genes, we hypothesize that anti-atherogenic (PS) and pro-atherogenic (OS) flow patterns induce distinct patterns of miRs, and hence the differential gene expressions and functional consequences. We will use in vitro, in vivo, and in silico approaches to develop an integrated system to elucidate the roles of miRs in regulating EC functions in response to different flow patterns. This multi-P.I. research project, by combining experimental data obtained from cultured ECs and mouse models with molecular, genomics and systems approaches, will elucidate the mechanisms of functional regulation by miRs in ECs under flows. In order to test our hypothesis, we propose the following five specific aims: (1) to establish miR expression profiles in cultured ECs in response to PS vs. OS. (2) To determine the target mRNAs of miRs in response to PS vs. OS. (3) To decipher the functional gene expression profiles regulated by miRs under PS vs. OS. (4) To elucidate the functional consequences of miR regulation under PS vs. OS. (5) To verify the role of miRs in functional regulation of vascular ECs exposed to different flow patterns in vivo. In this proposal the role of miR in regulating vascular functions will be studied under different flow patterns with a combination of experimental and computational approaches to perform multi-scale analyses from miRs/mRNAs to cellular functions. This innovative, multidisciplinary project includes (a) comprehensive genome-wide approaches to establish the miR profiles in ECs, (b) CLIP-seq approaches to elucidate the interactions between miRs and target mRNAs, (c) systems biology approaches to map the functional gene expression and biological consequence regulated by miRs, and (d) in vivo approaches in lesion-induction mice to validate the roles of miRs under different flow patterns established in vitro. The results will enhance the mechanistic insights of the roles of mechano- regulation and functional genomics at the systems biology level and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in the regulation of mRNA stability and translational repression, leading to the modulation of ~30% of the human genome. We will use in vitro, in vivo, and in silico technologies to develop an integrated systems approach to elucidate the roles of miRs in regulating endothelial functions in normal and pathophysiological flow conditions. The result may and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases.

DESCRIPTION (provided by applicant): The focal nature of the atherosclerotic lesions indicates that hemodynamic forces are critical for the regulation of vascular homeostasis in health and disease. Responses of vascular endothelial cells (ECs) to hemodynamic forces play significant roles in such regulations. In vivo studies implicate that the ECs in branch points express pro-atherogenic phenotypes. In contrast, ECs in the straight parts of the arterial tree are exposed to high shear flow with a large net forward direction, and these regions are generally spared from atherosclerosis. The in vitro studies by others and us suggest that steady and pulsatile shear stresses (PS) with a net forward direction, which simulates the flow condition at the straight part of the arterial tree, induce genes involved in anti-proliferation, anti-oxidation anti-inflammation, and maintenance of vascular tone, with athero-protective effects such as reduction of cell turnover, prevention of white cell recruitment, promotion of wound healing, and adaptive remodeling. In contrast, oscillatory shear stress (OS) without a significant forward direction is atherogenic by activating pro-proliferative, pro-oxidative, and pro-inflammatory genes. We hypothesize that, while PS and OS may activate similar signaling events at the initial stage, the results will diverge with time. Time-dependent mapping of the signal networks will lead to temporal resolution of the gene expression profiles, hence the differential functional consequences of PS vs. OS. In this proposed project, we will examine the signaling, transcriptional regulations, and functional phenotypes of ECs under PS and OS over time. Mapping the differential pathways under these flow conditions requires the use of systems biology approaches that provides a comprehensive mechanistic and network perspective on the diferential responses to stresses. In order to systematically map the flow-regulation of EC functions, we propose the following specific aims: (1) To establish the temporal map of EC signaling events under PS vs, OS. (2) To investigate the transcriptional regulations of EC gene expression under PS vs, OS. (3) To examine the temporal resolution of phenotypic responses of ECs under PS vs, OS. (4) To integrate molecular events and EC functions by reconstruction of signaling models. (5) To validate the defined EC signaling events and gene expressions in mouse arterial tree. Under these Specific Aims, we will conduct experiments systematically to obtain the data necessary for the systems biology analyses to construct the molecular and pathway models for the physiological and pathological regulations of EC molecular events and functional consequences. This integrative and collaborative systems biology approach will generate new insights into the intricate process of mechanotransduction by which different flow patterns modulate homeostasis in the arterial wall. These findings will greatly enhance our understanding of the molecular and mechanical bases of atherosclerosis, a major pathophysiological event in cardiovascular diseases.

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Ranging from 18 to 24 nt (22 nt in general), miRNAs regulate gene expression by suppressing protein translation of target mRNAs or enhancing their degradation. miRNAs targeting mRNAs depends on the association of the miRNA/mRNA complex with Argonaute (Ago) endonuclease to form the miRNA-induced silencing complex (miRISC). We used sequencing by synthesis (SBS) deep sequencing to study the miRNAs in cultured vascular endothelial cells (ECs) exposed to hypoxia. Bioinformatics approaches were then used to analyze the hypoxia-responsive miRNAs at the genome-wide scale. Among miRNAs with greatly changed expression, Let-7s and miR-103/107 target Ago1. This result suggests that an miRNA-mediated translational de-suppression mechanism may occur in ECs under hypoxia. With these data acquired from high-throughput screening and in silico approaches, two specific aims are proposed to test the hypothesis that under hypoxia, the increased Let-7s and miR- 103/107 down-regulate Ago1. Such a suppression of Ago1 decreases miRISC-mediated miRNA targeting and hence up-regulates targeted mRNAs, which encode highly translated proteins in ECs responding to hypoxia, including vascular endothelial growth factor. Specific Aim 1 will study the Ago1-regulated miRNA/mRNA targeting in ECs responding to hypoxia. We will use Ago1 cross-linking immunoprecipitation sequencing (CLIP-seq) to profile the miRISC- associated miRNAs and their mRNA targets in ECs under normoxia and hypoxia. Specific Aim 2 will decipher the functional consequences of the miRNA-mediated translational de- suppression in cultured ECs and mouse hindlimb. Specifically, we will manipulate the expression of Let-7s and miR-103/107 in vitro and in vivo. The miRISC-mediated miRNA/mRNA targeting, mRNA-encoded proteins, and angiogenesis under normoxia and hypoxia will be examined. The elucidated mechanism will reveal mechanistic insights underlying the miRNA-regulated gene expression in ECs in response to hypoxia.

DESCRIPTION (provided by applicant): Extracellular microRNAs (miRs) in endothelial cells (ECs) can be transported by extracellular carriers to regulate the gene expression in the recipient cells, i.e., smooth muscle cells (SMCs) and macrophages (MOs), and hence modulate vascular physiology and pathophysiology. In the current funding cycle, we demonstrated that distinct flow patterns, i.e. pulsatile shear (PS, the main feature of atheroprotective flow) and oscillatory shear (OS, the main feature of atheroprone flow) differentially regulate the miR expression profiles (miRomes) in ECs to cause up- or down-regulation of genes involved in inflammation, redox state, and proliferation. Moreover, others and we have shown that the shear stress-modulated miRs in ECs (e.g., miR-126 and miR- 143/145) are transferable via carriers such as exosomes or Ago2 to SMCs to target their gene expression and alter their phenotype. In relation to translational application, patients with cardiovascular diseases have been shown to have elevations of miR-126 and miR-92a in association with extracellular vesicles, and a decrease of their associations with lipoproteins. These findings led to the hypothesis of this new proposal that atheroprotective and atheroprone flow patterns modulate distinct miR-transportomes and regulate different miR-targetomes to result in beneficial or detrimental outcome of the vasculature. Specifically, we will expand our study to investigate the mechanisms and functional consequences of the shear stress-regulated EC miR transportomes and SMC/MO targetomes. The four Specific Aims are: (1) to investigate the EC miR transportomes regulated by PS vs. OS. (2) To elucidate the miR uptake mechanism and miR targetomes in recipient SMCs and MOs under PS and OS. (3) To determine the roles of PS- and OS-modulated miR transportomes and targetomes in regulating vascular functions. (4) To validate the flow regulation of miR expression and transmission in the vasculature in animal studies in vivo and in clinical samples from patients with cardiovascular diseases. We will perform flow channel experiments, high-throughput screening, and systems biology analysis and modeling to elucidate the diverse transportomes regulated by PS vs. OS in modulating the targetomes and functions of SMCs and MOs. We will validate and translate our in vitro and in silico results by using mouse models as well as human clinical samples. The proposed research aims at investigating the mechanisms by which miRs mediate the flow-regulation of vascular phenotypes and functions, with the ultimate goal of elucidating the interplays of miRs and shear pattern in the regulation of vascular homeostasis for translational applications.

Project Summary Atheroprone flow-induced endothelial dysfunction, characterized by enhanced inflammatory response, is an early vascular event leading to the focal distribution of atherosclerosis. Despite the causality between atheroprone flow pattern and endothelial dysfunction, key molecular events linking mechanical stimuli to the pro-inflammatory phenotype of vascular endothelial cells (ECs) and the consequent susceptibility to atherogenesis remain poorly understood. We recently demonstrated that atheroprone flow activates sterol regulatory element binding protein 2 (SREBP2) in ECs, which induces the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through transcriptional induction (i.e., Signal 1) and higher-order assembly (i.e., Signal 2). Such unresolved EC innate immune response leads to endothelial dysfunction and ensuing atherosclerosis. We have also identified that atheroprone flow induces TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA). Newly performed experiments indicate that SREBP2 transcriptionally upregulates TIFA and inflammasome components (Signal 1), and that phosphorylated TIFA Threonine-9 (TIFA pT9) oligomerizes TIFA leading to the higher-order assembly of NLRP3 inflammasome (Signal 2). These observations provide the foundation for the guiding hypothesis that atheroprone flow, through transcriptional induction and post-translational modification of TIFA, activates NLRP3 inflammasome in the endothelium, which contributes to atherosclerosis susceptibility. Three Specific Aims are proposed to test this hypothesis. Specific Aim 1 will elucidate the molecular mechanism by which atheroprone flow primes the induction of the NLRP3 inflammasome through Signal 1. In vitro flow channel experiments with oscillatory shear stress (OS) simulating atheroprone flow will be used to elucidate the transcriptional cascade of SREBP2-TIFA-NLRP3 inflammasome that respond to atheroprone flow. Specific Aim 2 will delineate the molecular basis by which atheroprone flow activates the NLRP3 inflammasome via Signal 2. Specifically, mechanobiology and FRET-based bioimaging will be used to investigate the role of TIFA pT9 in the higher-order assembly of NLRP3 inflammasome in ECs in response to atheroprone flow. Specific Aim 3 will investigate the role of atheroprone flow-activated SREBP2-TIFA-NLRP3 inflammasome in endothelial dysfunction, leading to atherosclerosis in vivo. We will examine endothelial function, vascular inflammation, and atherosclerosis in EC-SREBP2-/-, TIFA-/-, and TIFA T9A knock-in mice with or without an ApoE-/- background. The proposed experimental approaches that combine vascular, mechanobiological, biosensor, and animal experiments will systemically analyze the mechano and molecular basis of atheroprone flow-induced innate immune response in ECs and its functional relevance to atherosclerosis susceptibility.

More than 25% of children with Kawasaki disease (KD), the most common cause of pediatric acquired heart disease, develop coronary artery abnormalities (CAAs) despite standard therapy with intravenous immunoglobulin. Once aneurysms have formed, the damage to the arterial wall is irreversible, such as stenoses and calcification that may lead to ischemic complications. This collaborative team has established that activation of the IL-1 pathway and endothelial-to-mesenchymal transition (EndoMT) with downregulation of krüppel-like factor 4 (KLF4) are key to the pathogenesis of KD vasculitis. Our novel discovery of the central role of TIFA (TNF receptor-associated factor-interacting protein with a forkhead-associated domain) in NLRP3 inflammasome activation in endothelial cell (EC) injury is proposed as a further mechanism of KD vasculitis. We propose a bold plan to unite this research team with expertise in vascular biology, immunology, and clinical KD to unravel the mechanisms underlying vascular injury in KD and pilot a novel therapeutic approach. The guiding hypothesis is that acute KD not only activates the IL-1/TIFA/NLRP3 inflammasome in the endothelium, but also reduces EC homeostasis, both of which are critical in the pathogenesis of KD. The blockade of the IL-1-dependent innate immune response by anakinra (recombinant IL-1R antagonist) in combination with the restoration of EC function by atorvastatin will reduce vascular damage in acute KD. Three specific aims are proposed to test this hypothesis. Specific Aim 1 will delineate the molecular basis by which anakinra blocks activation of the innate immune response associated with KD vasculitis. In vitro, ex vivo, and in vivo experiments will investigate the role of IL-1, TIFA, and NLRP3 in KD-mediated EC dysfunction and vasculitis using pathway-specific siRNA knockdown, recombinant overexpression, pharmacologic agonists and inhibitors (including anakinra), as well as loss-of- function mice. Specific Aim 2 will elucidate the molecular basis by which atorvastatin restores EC function in acute KD. Mouse lines with gain- and loss-of-function of KLF4 as well as lineage tracing experiments will be performed to decipher the molecular mechanisms underlying EC dysfunction in KD vasculopathy. Specific Aim 3 will determine the synergistic effect of anakinra/atorvastatin combination therapy in LCWE-injected mice and in KD patients. LCWE mouse models, including Tifa-/- and Klf4-/-, will be utilized to test the effect of anakinra/atorvastatin combination therapy on vascular inflammation and EC function. Furthermore, acute KD patients with early CAAs will be enrolled in a pilot study of anakinra/atorvastatin combination therapy. Cytokine levels and MMP activity in sera and transcript abundance in whole blood RNA will be measured pre- and post- treatment. These sera will also be tested in vitro with cultured ECs with read-outs for EC innate immune responses and EC dysfunction. The synergistic expertise of the three teams in this multi-PI proposal provides a unique opportunity to understand molecular mechanisms underlying KD vasculopathy and rapidly translate the findings into a pilot study in children with in vitro and in vivo assessments of the treatment effect.

The focal distribution of atherosclerotic lesions in the arterial tree is attributable to the exposure of the vascular endothelial cells (ECs) to the atheroprone oscillatory shear (OS) at vessel branch points and curvatures, in contrast to the atheroprotective pulsatile shear (PS) at straight parts of the vessel. These two types of flow patterns induce distinct expression profiles of miRNAs (miRs) that differentially regulate the expression of target geness, resulting in distinct functional outcomes. Nucleolin (NCL) is a multi-functional protein that plays critical roles in regulating cell functions through epigenetic, transcriptional, post-transcriptional, and translational regulations. We propose that NCL regulates miRome (miR transcription and maturation) through its interactions with DNA (via g-quadruplex sequences) and RNA (via UCCCGA consensus sequence). Our preliminary results have demonstrated that PS induces the phosphorylation of NCL that leads to an increased binding of NCL to the promoter/enhancer regions of miR23b/27b to increase miR transcription. On the other hand, under OS, the non- phosphorylated NCL binds to miR93/miR484 and Drosha to facilitate miR maturation. These novel findings have led to the hypothesis of this renewal proposal: NCL is differentially regulated by atheroprotective and atheroprone flow patterns to modulate the miRome at transcriptional and maturation levels, thus playing an important role in vascular homeostasis in health and disease. To test this hypothesis, we propose the following four Specific Aims: 1) to examine the molecular mechanisms underlying PS- vs. OS-regulation of NCL in miR transcriptome, 2) to determine the molecular mechanisms underlying PS- vs. OS-regulation of NCL- modulated miR maturation, 3) to integrate the multi-layer regulations of miRome by NCL and the functional consequences under PS and OS with the use of systems approaches, 4) to validate the role of NCL in regulating vascular function in vivo. In the proposed research, we will use a combination of in vitro endothelial cell biology, in vivo mouse models, in silico network construction/data mining, and clinical samples derived from patients with cardiovascular diseases to decipher the mechanisms of flow modulation of NCL-regulation of miRome. The findings will result in novel understanding of the role of NCL in regulating vascular functions in health and disease via miR regulations. !

Project Summary In the wake of COVID-19 pandemic, fever with severe systemic inflammation and shock, known as Pediatric Inflammatory Multisystem Syndrome (PIMS), has evolved as a new threat to children. PIMS was originally reported in Western Europe and the number of cases is rapidly increasing in the U.S. A hallmark of PIMS has been heart failure leading to shock and the absence of significant pulmonary disease. The clinical presentation in these patients shares many features with Kawasaki disease (KD), the most common cause of acquired heart disease in children, which itself can present with distributive shock requiring inotropic and vasoactive support in the intensive care unit. Several PIMS patients are either SARS-CoV-2 PCR positive or have developed antibodies against SARS-CoV-2, suggesting that PIMS is an immune-mediated reaction to antecedent exposure to the virus. Curiously, at the same time that patients are being diagnosed with PIMS, the numbers of children with typical KD has increased dramatically in these same regions. The emergence of PIMS is so new and so rapidly evolving that there are literally no publications, treatment guidelines or clinical trials related to these seriously affected pediatric patients. Through our network of 30 pediatric centers participating in KIDCARE, our comparative effectiveness trial for treatment-resistant KD funded by PCORI, we are collecting patient data and clinical samples to support the work proposed here. The goal of this administrative supplement is to analyze demographic, clinical and laboratory data in conjunction with assays using patient cells and sera, which will allow us to study the relationships among SARS-CoV-2 infection, typical KD, and PIMS and to model different therapeutic strategies against PIMS. Three specific aims are proposed to achieve this goal. Specific Aim 1 will profile and compare clinical features of PIMS and typical KD. Demographic and clinical data and the neutrophil response to intravenous immunoglobulin (IVIG) will be compared between these two illnesses. Cytokine profiles and inflammatory markers in plasma from acute and subacute PIMS and typical KD will also be compared. Specific Aim 2 will elucidate molecular features of PIMS and compare with typical KD. We will use RNA-seq, ELISA, and Western blot analyses to profile changes in levels of molecules related to inflammation (e.g., TIFA, NFk?, NLRP3-inflammasome, IL-1, IL-6, TNF?) and cardiovascular health (KLF4, miR-483, ACE2) in endothelial cells and cardiomyocytes. Specific Aim 3 will test the efficacy of drug therapy for PIMS by comparing the in vitro effects of intravenous immunoglobulin, steroids, and anakinra on inflammatory pathways and cardiovascular biomarkers in endothelial cells and cardiomyocytes treated with sera from acute PIMS patients prior to therapy. The synergistic expertise of the investigative teams in this multi-PI supplement provides a unique opportunity to understand the clinical features, molecular basis, and efficacy of drug treatment of PIMS as compared to KD.

Project Abstract ? MAE-WEST SCORE Project 3 Over the course of life, chronic stressors contribute to multi-organ aging and dysfunction and, ultimately, the development of clinical disease. Sex remains a critical determinant of the nature and pace of aging and ultimately longevity. Among mammalian species, it is even more clear that females fundamentally age differently from males. With advancing chronologic age in humans, differences in biological aging between women and men become even more pronounced, culminating in the female predominance for a number of important morbid disease conditions, including notably Alzheimer?s disease and related dementias (ADRD), heart failure with preserved ejection fraction (HFpEF), progressive chronic kidney disease (CKD), and in turn systemic frailty. Mechanisms underlying the female predominance for these major morbidities remains unknown and are not explained by variations in sex hormones or survival bias. Our preliminary work supports a central hypothesis that sexual dimorphism in inflammatory eicosanoid mediators contribute to sex differences in microvascular dysfunction and, in turn, to sex differences in age-related multi-organ disease, including for ADRD, HFpEF and CKD. Elucidating a common pathophysiologic basis for the female predominance of ADRD, HFpEF, and CKD holds the key to effective interventions for reducing the excess burden of age-related disease in women. Motivated our findings and the critical need to understand the determinants and drivers of sex differences in major age-related disease outcomes, we propose to establish the Microvascular Aging and Eicosanoids ? Women?s Evaluation of Systemic aging Tenacity (MAE-WEST) (?You are never too old to become younger!?) Specialized Center of Research Excellence (SCORE) on Sex Differences, in response to NIH RFA-OD-19-013. Our goal is to form a robust and sustainable structure of academic activities centered on systematically interrogating sex differences in the relationship among eicosanoids, microvascular dysfunction, and age-related end-organ disease, with an initial focus on the microvascular aging effects on brain, heart, and kidney function. This goal will be achieved by an outstanding collaborative team of clinician-scientists (with expertise in geriatrics, cardiology, and nephrology), epidemiologists, basic and translational scientists, analytical chemists, biostatisticians, and bioinformaticians. Leveraging our collective experience, resources, and infrastructure, we will advance the scientific enterprise through 3 foundational projects aligned and complementary yet independent. Project 3 will determine the causal role of sex-specific eicosanoids in receptor activation, endothelial cell dysfunction and microvascular aging in experimental models and human endothelial cells. This basic science project will establish a direct, causal role for sex-specific eicosanoids on endothelial cell activation in vitro and microvascular dysfunction in experimental models. Using forward genetics, we will determine the specific receptors that mediate the effects of sex-specific eicosanoids in human endothelial cells.