CRISPR screening uncovers a central requirement for HHEX in pancreatic lineage commitment and plasticity restriction.

The pancreas and liver arise from a common pool of progenitors. However, the underlying mechanisms that drive their lineage diversification from the foregut endoderm are not fully understood. To tackle this question, we undertook a multifactorial approach that integrated human pluripotent-stem-cell-guided differentiation, genome-scale CRISPR-Cas9 screening, single-cell analysis, genomics and proteomics. We discovered that HHEX, a transcription factor (TF) widely recognized as a key regulator of liver development, acts as a gatekeeper of pancreatic lineage specification. HHEX deletion impaired pancreatic commitment and unleashed an unexpected degree of cellular plasticity towards the liver and duodenum fates. Mechanistically, HHEX cooperates with the pioneer TFs FOXA1, FOXA2 and GATA4, shared by both pancreas and liver differentiation programmes, to promote pancreas commitment, and this cooperation restrains the shared TFs from activating alternative lineages. These findings provide a generalizable model for how gatekeeper TFs like HHEX orchestrate lineage commitment and plasticity restriction in broad developmental contexts.

Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells.

Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a "safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies.

Heparan sulfate proteoglycan, integrin, and syndecan-4 are mechanosensors mediating cyclic strain-modulated endothelial gene expression in mouse embryonic stem cell-derived endothelial cells.

It is widely believed that the differentiation of embryonic stem cells (ESCs) into viable endothelial cells (ECs) for use in vascular tissue engineering can be enhanced by mechanical forces. In our previous work, we reported that shear stress enhanced important EC functional genes on a CD31+ /CD45- cell population derived from mouse ESC committed to the EC lineage. In the present study, in contrast to the effects of shear stress on this cell population, we observed that cyclic strain significantly reduced the expression of EC-specific marker genes (vWF, VE-cadherin, and PECAM-1), tight junction protein genes (ZO-1, OCLD, and CLD5), and vasoactive genes (eNOS and ET1), while it did not alter the expression of COX2. Taken together, these studies indicate that only shear stress, not cyclic strain, is a useful mechanical stimulus for enhancing the properties of CD31+ /CD45- cells for use as EC in vascular tissue engineering. To begin examining the mechanisms controlling cyclic strain-induced suppression of gene expression in CD31+ /CD45- cells, we depleted the heparan sulfate (HS) component of the glycocalyx, blocked integrins, and silenced the HS proteoglycan syndecan-4 in separate experiments. All of these treatments resulted in the reversal of cyclic strain-induced gene suppression. The current study and our previous work provide a deeper understanding of the mechanisms that balance the influence of cyclic strain and shear stress in endothelial cells.

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic ß-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development.

Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive ß-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.

Heparan sulfate proteoglycans mediate renal carcinoma metastasis.

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive tumor cells with high metastatic potential have enhanced invasion rates in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) in the glycocalyx plays an important role in this flow mediated mechanostransduction and upregulation of invasive and metastatic potential. In this study, highly metastatic renal cell carcinoma cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chamber and microfluidic assays, we show that flow-enhanced invasion is suppressed in HS deficient cells. To assess the ability of these cells to metastasize in vivo, parental or knockdown cells expressing fluorescence reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from the NDST1 knockdown cells compared to control cells with intact HS. The ability of these cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx in tumor progression.

A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells.

The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.

Fluid Mechanics, Arterial Disease, and Gene Expression.

This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.

Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion.

Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) was investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiological levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP activity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx - a potential target for therapeutics.

Homologous recombination DNA repair genes play a critical role in reprogramming to a pluripotent state.

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

Effect of the glycocalyx layer on transmission of interstitial flow shear stress to embedded cells.

In this paper, a simple theoretical model is developed to describe the transmission of force from interstitial fluid flow to the surface of a cell covered by a proteoglycan / glycoprotein layer (glycocalyx) and embedded in an extracellular matrix. Brinkman equations are used to describe flow through the extracellular matrix and glycocalyx layers and the solid mechanical stress developed in the glycocalyx by the fluid flow loading is determined. Using reasonable values for the Darcy permeability of extracellular matrix and glycocalyx layers and interstitial flow velocity, we are able to estimate the fluid and solid shear stresses imposed on the surface of embedded vascular, cartilage and tumor cells in vivo and in vitro. The principal finding is that the surface solid stress is typically one to two orders of magnitude larger than the surface fluid stress. This indicates that interstitial flow shear stress can be sensed by the cell surface glycocalyx, supporting numerous recent observations that interstitial flow can induce mechanotransduction in embedded cells. This study may contribute to understanding of interstitial flow-related mechanobiology in embryogenesis, tumorigenesis, tissue physiology and diseases and has implications in tissue engineering.

Enhanced osteogenesis of human mesenchymal stem cells by periodic heat shock in self-assembling peptide hydrogel.

The mechanisms for the heat-induced osteogenesis are not completely known and the thermal regulation of human mesenchymal stem cell (hMSC) differentiation is not well studied. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into osteoblasts in self-assembling peptide hydrogel and on tissue culture plates were investigated. hMSCs isolated from human bone marrow were seeded in conventional culture plates (two-dimensional [2D] culture) and on the surface of three-dimensional (3D) PuraMatrix peptide hydrogel (3D culture), followed by 1 h HS at 41°C once a week during osteogenic differentiation. Alkaline phosphatase (ALP) activity was enhanced in both 2D and 3D cultures via periodic HS at early stage of differentiation; meanwhile, HS significantly increased the calcium deposition at day 19 and 27 of differentiation in both 2D and 3D cultures. The periodic HS also upregulated osteo-specific genes, osterix (OSX) on day 11, osteopontin (OP) on day 19, and bone morphogenetic protein 2 (BMP2) on day 25 in 2D culture. In 3D PuraMatrix culture, the runt-related transcription factor 2 (Runx2) was upregulated by HS on day 25 of differentiation. The heat shock protein 70 (HSP70) was significantly upregulated by HS in differentiated hMSCs analyzed at 24 h after HS. These results demonstrate that HS induced an earlier differentiation of hMSCs and enhanced the maturation of osteoblasts differentiated from hMSCs. Therefore, mild HS treatment may be potentially used to enhance the bone regeneration using hMSCs. Our data will guide the design of in vivo heating protocols and enable further investigations in thermal treatments of MSC osteogenesis for bone tissue engineering.

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells.

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression.

BACKGROUND: Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate. METHODOLOGY/PRINCIPAL FINDINGS: A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs. CONCLUSIONS/SIGNIFICANCE: Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

Fluid flow mechanotransduction in vascular smooth muscle cells and fibroblasts.

Understanding how vascular wall endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs) sense and transduce the stimuli of hemodynamic forces (shear stress, cyclic strain, and hydrostatic pressure) into intracellular biochemical signals is critical to prevent vascular disease development and progression. ECs lining the vessel lumen directly sense alterations in blood flow shear stress and then communicate with medial SMCs and adventitial FBs to regulate vessel function and disease. Shear stress mechanotransduction in ECs has been extensively studied and reviewed. In the case of endothelial damage, blood flow shear stress may directly act on the superficial layer of SMCs and transmural interstitial flow may be elevated on medial SMCs and adventitial FBs. Therefore, it is also important to investigate direct shear effects on vascular SMCs as well as FBs. The work published in the last two decades has shown that shear stress and interstitial flow have significant influences on vascular SMCs and FBs. This review summarizes work that considered direct shear effects on SMCs and FBs and provides the first comprehensive overview of the underlying mechanisms that modulate SMC secretion, alignment, contraction, proliferation, apoptosis, differentiation, and migration in response to 2-dimensional (2D) laminar, pulsatile, and oscillating flow shear stresses and 3D interstitial flow. A mechanistic model of flow sensing by SMCs is also provided to elucidate possible mechanotransduction pathways through surface glycocalyx, integrins, membrane receptors, ion channels, and primary cilia. Understanding flow-mediated mechanotransduction in SMCs and FBs and the interplay with ECs should be helpful in exploring strategies to prevent flow-initiated atherosclerosis and neointima formation and has implications in vascular tissue engineering.

Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.

BACKGROUND: Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D) environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP) expression in rat vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D. METHODOLOGY/PRINCIPAL FINDINGS: Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS) chains from proteoglycan (PG) core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1) suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13) expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK) also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs) were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity. CONCLUSIONS/SIGNIFICANCE: We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction mechanism via HSPG-mediated FAK activation in 3D. This study will be of interest in understanding the flow-related mechanobiology in vascular lesion formation, tissue morphogenesis, cancer cell metastasis, and stem cell differentiation in 3D, and also has implications in tissue engineering.

Shear stress modulation of smooth muscle cell marker genes in 2-D and 3-D depends on mechanotransduction by heparan sulfate proteoglycans and ERK1/2.

BACKGROUND: During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Exposure to 8 dyn/cm(2) laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of alpha-smooth muscle actin (alpha-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH(2)O, approximately 0.05 dyn/cm(2), 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of alpha-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of alpha-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs). CONCLUSIONS/SIGNIFICANCE: The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.

Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies.

The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L (p)), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L (p) and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

Interstitial flow induces MMP-1 expression and vascular SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism.

The migration of vascular smooth muscle cells (SMCs) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have previously shown that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression, which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor PD-98059 completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation, whereas PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of activator protein-1 transcription factors (c-Jun and c-Fos), whereas PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, whereas silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggest that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.