Suli Yuan

Mechanotransduction in Response to Shear Stress ROLES OF RECEPTOR TYROSINE KINASES, INTEGRINS, AND Shc

Shear stress, the tangential component of hemodynamic forces, activates many signal transduction pathways in vascular endothelial cells. The conversion of mechanical stimulation into chemical signals is still unclear. We report here that shear stress (12 dynes/cm2) induced a rapid and transient tyrosine phosphorylation of Flk-1 and its concomitant association with the adaptor protein Shc; these are accompanied by a concurrent clustering of Flk-1, as demonstrated by confocal microscopy. Our results also show that shear stress induced an association of αvβ3 and β1 integrins with Shc, and an attendant association of Shc with Grb2. These associations are sustained, in contrast to the transient Flk-1·Shc association in response to shear stress and the transient association between αvβ3 integrin and Shc caused by cell attachment to substratum. Shc-SH2, an expression plasmid encoding the SH2 domain of Shc, attenuated shear stress activation of extracellular signal-regulated kinases and c-Jun N-terminal kinases, and the gene transcription mediated by the activator protein-1/12-O-tetradecanoylphorbol-13-acetate-responsive element complex. Our results indicate that receptor tyrosine kinases and integrins can serve as mechanosensors to transduce mechanical stimuli into chemical signals via their association with Shc.

Shear Stress Activates p60src-Ras-MAPK Signaling Pathways in Vascular Endothelial Cells

The aim of this study was to elucidate the upstream signaling mechanism that mediates the fluid shear stress activation of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs), in vascular endothelial cells (ECs). Our results indicate that p60src is rapidly activated by fluid shear stress in bovine aortic endothelial cells (BAECs). Shear stress induction of the hemagglutinin (HA) epitope-tagged HA-JNK1 and the Myc epitope-tagged Myc-ERK2 was significantly attenuated by v-src(K295R) and c-src(K295R), the kinase-defective mutants ofv-src and c-src, respectively. HA-JNK1 and Myc-ERK2 were activated by c-src(F527), a constitutively activated form of p60src, and the activation was abolished by RasN17, a dominant-negative mutant of p2lras. In contrast, although HA-JNK1 and Myc-ERK2 were also activated by RasL61, an activated form of p21ras, the activation was not affected by v-src(K295R). These results indicate that p60src is upstream to the Ras-JNK and Ras-ERK pathways in response to shear stress. The shear stress inductions of the promoters of monocyte chemotactic protein-1 (MCP-1) and c-fos, driven by TPA-responsive element (TRE) and serum-responsive element (SRE), respectively, were attenuated by v-src(K295R). This attenuation is associated with decreased transcriptional activities of c-Jun and Elk-1, the transcription factors targeting TRE and SRE, respectively. Thus, p60src plays a critical role in the shear stress activation of MAPK pathways and induction of Activating Protein-1 (AP- 1)/TRE and Elk-1/SRE-mediated transcription in ECs.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

Significance The great challenge to developing an in vitro liver model lies in the limitation of current approaches to recapitulate the sophisticated liver microenvironment contributed by the complex microarchitecture and diverse cell combination. We demonstrate an innovative advancement toward simulating natural complexity by integrating a rapid 3D bioprinting technology with tissue engineering to develop a microscale hepatic construct consisting of physiologically relevant hexagonal units of liver cells and supporting cells. The entire construct is fabricated within several seconds on minimal UV illumination. The model enables the structural and functional improvements of human induced pluripotent stem cell-derived hepatic progenitor cells and therefore can be used in early personalized drug screening and liver pathophysiology studies in vitro. The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

Effects of Flow Patterns on the Localization and Expression of VE-Cadherin at Vascular Endothelial Cell Junctions: In vivo and in vitro Investigations

Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to fluctuating shear stress and have high endothelial permeability. Endothelial permeability is modulated by intercellular adhesion molecules such as VE-cadherin. This study was designed to elucidate the effects of different flow patterns on the localization and expression of VE-cadherin in endothelial cells (ECs) both in vivo and in vitro. VE-cadherin staining at EC borders was much stronger in the descending thoracic aorta and abdominal aorta, where the pulsatile flow has a strong net forward component than in the aortic arch and the poststenotic dilatation site beyond an experimental constriction, where the flow near the wall is complex and reciprocating with little net flow. With the use of flow chambers the effects of pulsatile flow (12 ± 4 dyn/cm2 at 1 Hz) and reciprocating flow (0.5 ± 4 dyn/cm2 at 1 Hz) on VE-cadherin organization in endothelial monolayers were studied in vitro. VE-cadherin staining was continuous along cell borders in static controls. Following 6 h of either pulsatile or reciprocating flow, the VE-cadherin staining at cell borders became intermittent. When the pulsatile flow was extended to 24, 48 or 72 h the staining around the cell borders became continuous again, but the staining was still intermittent when the reciprocating flow was similarly extended. Exposure to pulsatile or reciprocating flow for 6 and 24 h neither change the expression level of VE-cadherin nor its distribution between membrane and cytosol fractions as determined by Western blot and compared with static controls. These findings suggest that the cell junction remodeling induced by different flow patterns may result from a redistribution of VE-cadherin within the cell membrane. Both the in vivo and in vitro data indicate that pulsatile and reciprocating flow patterns have different effects on cell junction remodeling. The lack of junction reorganization in regions of reciprocating flow in vivo and in vitro may provide a mechanistic basis for the high permeability and the preferential localization of atherosclerosis in regions of the arterial stress with complex flow patterns and fluctuating shear stress.

Signal Transduction in Matrix Contraction and the Migration of Vascular Smooth Muscle Cells in Three-Dimensional Matrix

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (–80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30–40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.

Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm2 for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.

Laminar flow induces endothelial cell growth arrest through p53 phosphorylation and stabilization

We investigated the role of p53 in mediating the laminar flow-induced growth arrest in endothelial cells (ECs). c-Jun N-terminal kinase (JNK) activity induced by laminar flow is able to phosphorylate p53, an event which leads to the stabilization of p53. We also found that laminar flow caused an increase in the protein level of the exogenous HA-p53 in ECs. This increased p53 level would suppress EC proliferation.

Growth arrest and DNA damage inducible gene 45 (GADD45) is induced by laminar shear stress as identified by using DNA microarray technology

Laminar shear stress inhibits endothelial cell proliferation in vitro. Using DNA microarray technology, we found that many genes involved in growth arrest were up-regulated by laminar shear stress, including the Growth Arrest and DNA Damage Inducible Gene 45 (GADD45). The mRNA and protein levels of GADD45 were both increased in response to laminar flow, as shown by Northern blot and Western blot, respectively. The induction of GADD45 promoter by laminar shear stress was regulated by the tumor suppressor gene p53. These results provide a molecular basis for the inhibition of endothelial cell proliferation by laminar shear stress.