Kuei-Chun Wang

Nanoparticle biointerfacing by platelet membrane cloaking

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

MicroRNA-21 targets peroxisome proliferators-activated receptor-α in an autoregulatory loop to modulate flow-induced endothelial inflammation

Adhesion of circulating monocytes to vascular endothelial cells (ECs) is a critical event leading to vascular inflammation and, hence, development of atherosclerosis. MicroRNAs (miRs) are a class of endogenous, highly conserved, noncoding small RNAs that play important roles in regulating gene expression and cellular function, as well as pathogenesis of atherosclerosis. Here, we showed that oscillatory shear stress (OSS) induces the expression of miR-21 at the transcriptional level in cultured human umbilical vein ECs via an increased binding of c-Jun, which is a component of transcription factor activator protein-1 (AP-1), to the promoter region of miR-21. OSS induction of miR-21 inhibited the translation, but not transcription, of peroxisome proliferators-activated receptor-α (PPARα) by 3′-UTR targeting. Overexpression of miR-21 up-regulated AP-1 activation, which was attenuated by exogenous expression of PPARα. OSS and overexpression of miR-21 enhanced the expression of adhesion molecules vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and the consequential adhesion of monocytes to ECs. Overexpression of PPARα significantly attenuated the AP-1–mediated miR-21 expression. These results demonstrate a unique mechanism by which OSS induces AP-1–dependent miR-21 expression, which directly targets PPARα to inhibit its expression, thereby allowing activation of AP-1 and the promotion of monocyte adhesion. Our findings suggest the presence of a positive feedback loop that enables the sustained induction of miR-21, thus contributing to the proinflammatory responses of vascular endothelium under OSS.

Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up- and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PS-induced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.

Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell–cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell–cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell–cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

Matrix Stiffness Regulates Endothelial Cell Proliferation through Septin 9

Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs) was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa) in comparison to those with low stiffness (LSG, 1.72 kPa). ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9), the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin αvβ3 caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.

Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis.

Significance Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are emerging as key regulators of cell fate in response to biochemical and biophysical cues, but their role in modulating vascular homeostasis in health and disease is not clearly understood. In studying the flow regulation of YAP/TAZ activities in vascular endothelial cells, we find that disturbed flow (without a clear direction), but not laminar flow (with a clear direction), activates YAP/TAZ to promote the atheroprone phenotypes (proliferation and inflammation). We also demonstrate that inhibition of YAP/TAZ attenuates the atheroprone phenotypes and lesion progression in atherosclerotic mice. These findings indicate that YAP/TAZ serve as important mechanotransducers in the disturbed flow-induced atherogenesis and provide insights to the development of therapeutic strategy for cardiovascular diseases. The focal nature of atherosclerotic lesions suggests an important role of local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechanotransduction and vascular homeostasis. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of atheroprone endothelial phenotypes and the consequential development of atherosclerotic lesions. We found that exposure of cultured endothelial cells (ECs) to the atheroprone disturbed flow resulted in YAP/TAZ activation and translocation into EC nucleus to up-regulate the target genes, including cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). In contrast, the athero-protective laminar flow suppressed YAP/TAZ activities. En face analysis of mouse arteries demonstrated an increased nuclear localization of YAP/TAZ and elevated levels of the target genes in the endothelium in atheroprone areas compared with athero-protective areas. YAP/TAZ knockdown significantly attenuated the disturbed flow induction of EC proliferative and proinflammatory phenotypes, whereas overexpression of constitutively active YAP was sufficient to promote EC proliferation and inflammation. In addition, treatment with statin, an antiatherosclerotic drug, inhibited YAP/TAZ activities to diminish the disturbed flow-induced proliferation and inflammation. In vivo blockade of YAP/TAZ translation by morpholino oligos significantly reduced endothelial inflammation and the size of atherosclerotic lesions. Our results demonstrate a critical role of the activation of YAP/TAZ by disturbed flow in promoting atheroprone phenotypes and atherosclerotic lesion development. Therefore, inhibition of YAP/TAZ activation is a promising athero-protective therapeutic strategy.

MicroRNA-23b Regulates Cyclin-Dependent Kinase–Activating Kinase Complex Through Cyclin H Repression to Modulate Endothelial Transcription and Growth Under Flow

Objective—The site-specificity of endothelial phenotype is attributable to the local hemodynamic forces. The flow regulation of microRNAs in endothelial cells (ECs) plays a significant role in vascular homeostasis and diseases. The objective of this study was to elucidate the molecular mechanism by which the pulsatile shear flow–induced microRNA-23b (miR-23b) exerts antiproliferative effects on ECs. Approach and Results—We used a combination of a cell perfusion system and experimental animals to examine the flow regulation of miR-23b in modulating EC proliferation. Our results demonstrated that pulsatile shear flow induces the transcription factor Krüppel-like factor 2 to promote miR-23b biosynthesis; the increase in miR-23b then represses cyclin H to impair the activity and integrity of cyclin-dependent kinase–activating kinase (CAK) complex. The inhibitory effect of miR-23b on CAK exerts dual actions to suppress cell cycle progression, and reduce basal transcription by deactivating RNA polymerase II. Whereas pulsatile shear flow regulates the miR-23b/CAK pathway to exert antiproliferative effects on ECs, oscillatory shear flow has little effect on the miR-23b/CAK pathway and hence does not cause EC growth arrest. Such flow pattern–dependent phenomena are validated with an in vivo model on rat carotid artery: the flow disturbance induced by partial carotid ligation led to a lower expression of miR-23b and a higher EC proliferation in comparison with the pulsatile flow regions of the unligated vessels. Local delivery of miR-23b mitigated the proliferative EC phenotype in partially ligated vessels. Conclusions—Our findings unveil a novel mechanism by which hemodynamic forces modulate EC proliferative phenotype through the miR-23b/CAK pathway.

LINC00341 exerts an anti-inflammatory effect on endothelial cells by repressing VCAM1

The long noncoding RNAs (lncRNAs), which constitute a large portion of the transcriptome, have gained intense research interest because of their roles in regulating physiological and pathophysiological functions in the cell. We identified from RNA-Seq profiling a set of lncRNAs in cultured human umbilical vein endothelial cells (HUVECs) that are differentially regulated by atheroprotective vs. atheroprone shear flows. Among the comprehensively annotated lncRNAs, including both known and novel transcripts, LINC00341 is one of the most abundant lncRNAs in endothelial cells. Moreover, its expression level is enhanced by atheroprotective pulsatile shear flow and atorvastatin. Overexpression of LINC00341 suppresses the expression of vascular cell adhesion molecule 1 (VCAM1) and the adhesion of monocytes induced by atheroprone flow and tumor necrosis factor-alpha. Underlying this anti-inflammatory role, LINC00341 guides enhancer of zest homolog 2, a core histone methyltransferase of polycomb repressive complex 2, to the promoter region of the VCAM1 gene to suppress VCAM1. Network analysis reveals that the key signaling pathways (e.g., Rho and PI3K/AKT) are co-regulated with LINC00341 in endothelial cells in response to pulsatile shear. Together, these findings suggest that LINC00341, as an example of lncRNAs, plays important roles in modulating endothelial function in health and disease.

RAP2 mediates mechanoresponses of the Hippo pathway

Mammalian cells are surrounded by neighbouring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes1. The Hippo pathway effectors YAP (also known as YAP1) and TAZ (also known as WWTR1) are regulated by mechanical cues and mediate cellular responses to ECM stiffness2,3. Here we identified the Ras-related GTPase RAP2 as a key intracellular signal transducer that relays ECM rigidity signals to control mechanosensitive cellular activities through YAP and TAZ. RAP2 is activated by low ECM stiffness, and deletion of RAP2 blocks the regulation of YAP and TAZ by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, which activates RAP2 through PDZGEF1 and PDZGEF2 (also known as RAPGEF2 and RAPGEF6). At low stiffness, active RAP2 binds to and stimulates MAP4K4, MAP4K6, MAP4K7 and ARHGAP29, resulting in activation of LATS1 and LATS2 and inhibition of YAP and TAZ. RAP2, YAP and TAZ have pivotal roles in mechanoregulated transcription, as deletion of YAP and TAZ abolishes the ECM stiffness-responsive transcriptome. Our findings show that RAP2 is a molecular switch in mechanotransduction, thereby defining a mechanosignalling pathway from ECM stiffness to the nucleus.The Ras-related GTPase RAP2 is a key intracellular signal transducer by which extracellular matrix rigidity controls mechanosensitive cellular activities through YAP and TAZ.

VAMP3 and SNAP23 mediate the disturbed flow-induced endothelial microRNA secretion and smooth muscle hyperplasia

Significance Vascular homeostasis is regulated by the interactions between endothelial cells (ECs) and smooth muscle cells (SMCs). Our previous study demonstrated that endothelial microRNA-126-3p conveys the EC-to-SMC signaling that is regulated by hemodynamic shear stress to ECs. Here we show that the mechanisms by which shear stress modulates microRNA secretion include the regulations on vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Endothelial inhibition of VAMP3/SNAP23 reduces microRNA-126-3p secretion and rescues the SMC phenotype altered by atherogenic shear. Our findings provide a mechanistic explanation of the noncoding RNA mediation of detrimental effects of oscillatory shear on vascular function and suggest therapeutic potential of targeting the endothelial vesicular transport system in the control of smooth muscle proliferative diseases. Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern–specificity of SNARE activation and its contribution to the miRNA-mediated EC–SMC communication.