Responses of vascular endothelial cells (ECs) to hemodynamic forces play significant roles in the regulation of vascular homeostasis. In vivo studies have shown that the ECs in branch points of the arterial tree are exposing to disturbed flow (DF) and express pro-inflammatory and pro-atherogenic phenotypes. In contrast, ECs in the straight part of the arterial tree are exposed to laminar shear flow (LF) and are generally spared from atherosclerosis. We hypothesize that atheroprone and atheroprotective flows activate ECs with differential spatiotemporal characteristics at subcellular levels to trigger different cellular responses. We propose to use genetically encoded biosensors based on fluorescent proteins (FPs) and fluorescence resonance energy transfer (FRET) to visualize molecular activities in individual live cells with unprecedented spatiotemporal resolution. We will study the signals relays across the plasma membrane, between neighboring cells, as well as intracellular cytosol-nuclei transitions to understand the temporal and spatial dynamics of mechanotransduction. In order to achieve effectiveness of the biosensor studies, we will incorporate a new mOrange2-mCherry FRET pair together with the CFP-YFP pair to simultaneously monitor two different molecular events in the same live cell. We will further integrate fluorescence lifetime imaging microscopy (FLIM) to simultaneously visualize multiple molecular signals across the plasma membrane, between cells, and inside the cell body, with the use of correlative FRET imaging microscopy (CFIM) developed in our labs.
Three specific aims are proposed: 1) To visualize the spatiotemporal mechanotransduction across the plasma membrane: the extracellular shear stress (shear sensors) and intracellular molecular signals (transmembrane TRPC6 and Src activities at different membrane microdomains) will be simultaneously monitored under different flows to elucidate the roles of microdomains and molecular elements at the plasma membrane. 2) To dissect the role of TRPC6 in the regulation of adherent junctions (AJs) under different flows: an -catenin biosensor will be used to monitor the mechanical tension at AJs and its interplays with extra-/inter-cellular calcium ion concentrations. 3) To decipher the membrane-cytosol-nucleus ERK signaling for MCP-1 gene regulation: differential flow-regulations of the cytosolic and nucleic ERK FRET biosensors will be determined to reconstruct the spatiotemporal activation map of ERK in relation to MCP-1 gene expression. The results obtained from these studies will allow us to generate spatiotemporal correlation maps of molecular transductions/interactions and assess the roles of membrane microdomains/elements in regulating these events. These findings will provide novel understanding of the spatiotemporal basis of the molecular and mechanical mechanisms of atherosclerosis, a major pathophysiological event in cardiovascular diseases.

Public Health Relevance

We propose to use genetically coded biosensors to monitor the temporal and spatial cellular responses regulated by hemodynamic forces. The resultant mechanistic and pathway models will provide critical information on the mechanisms of atherosclerosis, a major pathophysiological event in cardiovascular diseases. The knowledge will also provide guidance for novel designs to target disease prevention, treatment, and management.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL121365-03
Application #
8972030
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Gao, Yunling
Project Start
2013-12-20
Project End
2017-11-30
Budget Start
2015-12-08
Budget End
2016-11-30
Support Year
3
Fiscal Year
2016
Total Cost
$648,035
Indirect Cost
$229,947
Name
University of California San Diego
Department
Biomedical Engineering
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Yeh, Yi-Ting; Serrano, Ricardo; François, Joshua et al. (2018) Three-dimensional forces exerted by leukocytes and vascular endothelial cells dynamically facilitate diapedesis. Proc Natl Acad Sci U S A 115:133-138
Pan, Yijia; Yoon, Sangpil; Zhu, Linshan et al. (2018) Acoustic mechanogenetics. Curr Opin Biomed Eng 7:64-70
Limsakul, Praopim; Peng, Qin; Wu, Yiqian et al. (2018) Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface. Cell Chem Biol 25:370-379.e4
Pan, Yijia; Yoon, Sangpil; Sun, Jie et al. (2018) Mechanogenetics for the remote and noninvasive control of cancer immunotherapy. Proc Natl Acad Sci U S A 115:992-997
Peng, Qin; Lu, Shaoying; Shi, Yuxin et al. (2018) Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors. Proc Natl Acad Sci U S A 115:E11681-E11690
Yoon, Sangpil; Wang, Pengzhi; Peng, Qin et al. (2017) Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound. Sci Rep 7:5275
Vennin, Claire; Chin, Venessa T; Warren, Sean C et al. (2017) Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis. Sci Transl Med 9:
Wan, Qiaoqiao; TruongVo, ThucNhi; Steele, Hannah E et al. (2017) Subcellular domain-dependent molecular hierarchy of SFK and FAK in mechanotransduction and cytokine signaling. Sci Rep 7:9033
Sun, Jie; Lei, Lei; Tsai, Chih-Ming et al. (2017) Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions. Nat Commun 8:477
Seong, Jihye; Huang, Min; Sim, Kyoung Mi et al. (2017) FRET-based Visualization of PDGF Receptor Activation at Membrane Microdomains. Sci Rep 7:1593

Showing the most recent 10 out of 32 publications