Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

Multicellular dynamics on structured surfaces: Stress concentration is a key to controlling complex microtissue morphology on engineered scaffolds.

Tissue engineers have utilised a variety of three-dimensional (3D) scaffolds for controlling multicellular dynamics and the resulting tissue microstructures. In particular, cutting-edge microfabrication technologies, such as 3D bioprinting, provide increasingly complex structures. However, unpredictable microtissue detachment from scaffolds, which ruins desired tissue structures, is becoming an evident problem. To overcome this issue, we elucidated the mechanism underlying collective cellular detachment by combining a new computational simulation method with quantitative tissue-culture experiments. We first quantified the stochastic processes of cellular detachment shown by vascular smooth muscle cells on model curved scaffolds and found that microtissue morphologies vary drastically depending on cell contractility, substrate curvature, and cell-substrate adhesion strength. To explore this mechanism, we developed a new particle-based model that explicitly describes stochastic processes of multicellular dynamics, such as adhesion, rupture, and large deformation of microtissues on structured surfaces. Computational simulations using the developed model successfully reproduced characteristic detachment processes observed in experiments. Crucially, simulations revealed that cellular contractility-induced stress is locally concentrated at the cell-substrate interface, subsequently inducing a catastrophic process of collective cellular detachment, which can be suppressed by modulating cell contractility, substrate curvature, and cell-substrate adhesion. These results show that the developed computational method is useful for predicting engineered tissue dynamics as a platform for prediction-guided scaffold design. STATEMENT OF SIGNIFICANCE: Microfabrication technologies aiming to control multicellular dynamics by engineering 3D scaffolds are attracting increasing attention for modelling in cell biology and regenerative medicine. However, obtaining microtissues with the desired 3D structures is made considerably more difficult by microtissue detachments from scaffolds. This study reveals a key mechanism behind this detachment by developing a novel computational method for simulating multicellular dynamics on designed scaffolds. This method enabled us to predict microtissue dynamics on structured surfaces, based on cell mechanics, substrate geometry, and cell-substrate interaction. This study provides a platform for the physics-based design of micro-engineered scaffolds and thus contributes to prediction-guided biomaterials design in the future.

Spatial Heterogeneity of Invading Glioblastoma Cells Regulated by Paracrine Factors.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. Impact Statement We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo.

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Heterogeneous Glioma Cell Invasion Under Interstitial Flow Depending on Their Differentiation Status.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor. A deeper mechanistic understanding of the invasion of heterogeneous GBM cell populations is crucial to develop therapeutic strategies. A key regulator of GBM cell invasion is interstitial flow. However, the effect of an interstitial flow on the invasion of heterogeneous GBM cell populations composed of glioma initiating cells (GICs) and relatively differentiated progeny cells remains unclear. In the present study, we investigated how GICs invade three-dimensional (3D) hydrogels in response to an interstitial flow with respect to their differentiation status. Microfluidic culture systems were used to apply an interstitial flow to the cells migrating from the cell aggregates into the 3D hydrogel. Phase-contrast microscopy revealed that the invasion and protrusion formation of the GICs in differentiated cell conditions were significantly enhanced by a forward interstitial flow, whose direction was the same as that of the cell invasion, whereas those in stem cell conditions were not enhanced by the interstitial flow. The mechanism of flow-induced invasion was further investigated by focusing on differentiated cell conditions. Immunofluorescence images revealed that the expression of cell-extracellular matrix adhesion-associated molecules, such as integrin ß1, focal adhesion kinase, and phosphorylated Src, was upregulated in forward interstitial flow conditions. We then confirmed that cell invasion and protrusion formation were significantly inhibited by PP2, a Src inhibitor. Finally, we observed that the flow-induced cell invasion was preceded by nestin-positive immature GICs at the invasion front and followed by tubulin ß3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion. Impact statement A mechanistic understanding of heterogeneous glioblastoma cell invasion is crucial for developing therapeutic strategies. We observed that the invasion and protrusion formation of glioma initiating cells (GICs) were significantly enhanced by forward interstitial flow in differentiated cell conditions. The expression of integrin ß1, focal adhesion kinase, and phosphorylated Src was upregulated, and the flow-induced invasion was significantly inhibited by a Src inhibitor. The flow-induced heterogeneous cell invasion was preceded by nestin-positive GICs at the invasion front and followed by tubulin ß3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion.

Microfluidic Device Setting by Coculturing Endothelial Cells and Mesenchymal Stem Cells.

The construction of vascular networks is essential for developing functional organ/tissue constructs in terms of oxygen and nutrient supply. Although recent advances in microfluidic techniques have allowed for the construction of microvascular networks using microfluidic devices, their structures cannot be maintained for extended periods of time due to a lack of perivascular cells. To construct long-lasting microvascular networks, it is important that perivascular cells are present to provide structural support to vessels, because in vivo microvessels are covered by perivascular cells and stabilized. Here, we describe a microfluidic cell culture platform for the construction of microvascular networks with supportive perivascular cells. Our results showed that microvascular networks covered by pericyte-like perivascular cells formed in a microfluidic device and their structures were maintained for at least 3 weeks in vitro.

Control of vessel diameters mediated by flow-induced outward vascular remodeling in vitro.

Vascular networks consist of hierarchical structures of various diameters and are necessary for efficient blood distribution. Recent advances in vascular tissue engineering and bioprinting have allowed us to construct large vessels, such as arteries, small vessels, such as capillaries and microvessels, and intermediate-scale vessels, such as arterioles, individually. However, little is known about the control of vessel diameters between small vessels and intermediate-scale vessels. Here, we focus on vascular remodeling, which creates lasting structural changes in the vessel wall in response to hemodynamic stimuli, to regulate vessel diameters in vitro. The purpose of this study is to control the vessel diameter at an intermediate scale by inducing outward remodeling of microvessels in vitro. Human umbilical vein endothelial cells and mesenchymal stem cells were cocultured in a microfluidic device to construct microvessels, which were then perfused with a culture medium to induce outward vascular remodeling. We successfully constructed vessels with diameters of 40-150 µm in perfusion culture, whereas vessels with diameters of <20 µm were maintained in static culture. We also revealed that the in vitro vascular remodeling was mediated by NO pathways and MMP-9. These findings provide insight into the regulation of diameters of tissue-engineered blood vessels. This is an important step toward the construction of hierarchical vascular networks within biofabricated three-dimensional systems.

Air-pressure-driven Separable Microdevice to Control the Anisotropic Curvature of Cell Culture Surface.

We report on a novel microdevice to tune the curvature of a cell-adhering surface by controlling the air-pressure and micro-slit. Human aortic smooth muscle cells were cultured on demi-cylindrical concaves formed on a microdevice. Their shape-adapting behavior could be tracked when the groove direction was changed to the orthogonal direction. This microdevice demonstrated live observation of cells responding to dynamic changes of the anisotropic curvature of the adhering surface and could serve as a new platform to pursue mechanobiology on curved surfaces.

Balance of interstitial flow magnitude and vascular endothelial growth factor concentration modulates three-dimensional microvascular network formation.

Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an in vitro three-dimensional (3D) angiogenesis model in a microfluidic device. This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.

Self-organization of hepatocyte morphogenesis depending on the size of collagen microbeads relative to hepatocytes.

Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte-collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell-cell and cell-collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell-microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.

Construction of Continuous Capillary Networks Stabilized by Pericyte-like Perivascular Cells.

IMPACT STATEMENT: Construction of capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. However, it is not well understood how to construct stable capillary networks that maintain a luminal size similar to that of capillary structures in vivo (i.e., <10 µm diameter). In this study, we demonstrated the construction of stable capillary networks covered by pericyte-like perivascular cells using an in vitro 3D angiogenesis model by optimizing interactions between endothelial cells and perivascular cells. Our 3D angiogenesis model can be combined with 3D culture of epithelial cells in the context of vascularization of 3D tissue-engineered constructs.

Comparison of organ-specific endothelial cells in terms of microvascular formation and endothelial barrier functions.

Every organ demonstrates specific vascular characteristics and functions maintained by interactions of endothelial cells (ECs) and parenchymal cells. Particularly, brain ECs play a central role in the formation of a functional blood brain barrier (BBB). Organ-specific ECs have their own morphological features, and organ specificity must be considered when investigating interactions between ECs and other cell types constituting a target organ. Here we constructed angiogenesis-based microvascular networks with perivascular cells in a microfluidic device setting by coculturing ECs and mesenchymal stem cells (MSCs). Furthermore, we analyzed endothelial barrier functions as well as fundamental morphology, an essential step to build an in vitro BBB model. In particular, we used both brain microvascular ECs (BMECs) and human umbilical vein ECs (HUVECs) to test if organ specificity of ECs affects the formation processes and endothelial barrier functions of an engineered microvascular network. We found that microvascular formation processes differed by the source of ECs. HUVECs formed more extensive microvascular networks compared to BMECs while no differences were observed between BMECs and HUVECs in terms of both the microvascular diameter and the number of pericytes peripherally associated with the microvasculatures. To compare the endothelial barrier functions of each type of EC, we performed fluorescence dextran perfusion on constructed microvasculatures. The permeability coefficient of BMEC microvasculatures was significantly lower than that of HUVEC microvasculatures. In addition, there were significant differences in terms of tight junction protein expression. These results suggest that the organ source of ECs influences the properties of engineered microvasculature and thus is a factor to be considered in the design of organ-specific cell culture models.

Construction of sinusoid-scale microvessels in perfusion culture of a decellularized liver.

There is a great deal of demand for the construction of transplantable liver grafts. Over the last decade, decellularization techniques have been developed to construct whole liver tissue grafts as potential biomaterials. However, the lack of intact vascular networks, especially sinusoids, in recellularized liver scaffolds leads to hemorrhage and thrombosis after transplantation, which is a major obstacle to the development of transplantable liver grafts. In the present study, we hypothesized that both mechanical (e.g., fluid shear stress) and chemical factors (e.g., fibronectin coating) can enhance the formation of hierarchical vascular networks including sinusoid-scale microvessels. We demonstrated that perfusion culture promoted formation of sinusoid-scale microvessels in recellularized liver scaffolds, which was not observed in static culture. In particular, perfusion culture at 4.7 ml/min promoted the formation of sinusoid-scale microvessels compared to perfusion culture at 2.4 and 9.4 ml/min. In addition, well-aligned endothelium was observed in perfusion culture, suggesting that endothelial cells sensed the flow-induced shear stress. Moreover, fibronectin coating of decellularized liver scaffolds enhanced the formation of sinusoid-scale microvessels in perfusion culture at 4.7 ml/min. This study represents a critical step in the development of functional recellularized liver scaffolds, which can be used not only for transplantation but also for drug screening and disease-modeling studies. STATEMENT OF SIGNIFICANCE: Decellularized liver scaffolds are promising biomaterials that allow production of large-scale tissue-engineered liver grafts. However, it is difficult to maintain recellularized liver grafts after transplantation due to hemorrhage and thrombosis. To overcome this obstacle, construction of an intact vascular network including sinusoid-scale microvessels is essential. In the present study, we succeeded in constructing sinusoid-scale microvessels in decellularized liver scaffolds via a combination of perfusion culture and surface coating. We further confirmed that endothelial cells in decellularized liver scaffolds responded to flow-derived mechanical stress by aligning actin filaments. Our strategy to construct sinusoid-scale microvessels is critical for the development of intact vascular networks, and addresses the limitations of recellularized liver scaffolds after transplantation.

Reconstruction of Hepatic Tissue Structures Using Interstitial Flow in a Microfluidic Device.

Construction of three-dimensional (3D) hepatic tissue structures is important for in vitro tissue engineering of the liver, because 3D culture of hepatocytes is critical for the maintenance of liver-specific functions. Although conventional 3D culture methods are useful for constructing 3D hepatic tissue structures, the precise control of culture microenvironments is required to construct more physiological tissues in vitro. Recent advances in microfluidics technologies have allowed us to utilize microfluidic devices for hepatic cell culture, which opened the door for creating more physiological 3D culture models of the liver. Here, we describe the method for the construction of hepatic tissue structures using a microfluidic device which has a 3D gel region with adjacent microchannels. Primary rat hepatocytes are seeded into a microchannel in a microfluidic device. The cells are then cultured in interstitial flow conditions, which leads to the construction of 3D tissue structures.

Integration of neurogenesis and angiogenesis models for constructing a neurovascular tissue.

Neurovascular unit (NVU) is a basic unit in the brain, including neurons, glial cells, blood vessels and extracellular matrix. This concept implies the importance of a three-dimensional (3D) culture model including these cell types for investigating brain functions. However, little is known about the construction of an in vitro 3D NVU model. In the present study, we aimed at constructing 3D neurovascular tissues by combining in vitro neurogenesis and angiogenesis models using a microfluidic platform, which is a critical step toward the NVU construction in vitro. Three gel conditions, which were fibrin gel, fibrin-Matrigel mixed gel and fibrin-hyaluronan mixed gel, were investigated to optimize the gel components in terms of neurogenesis and angiogenesis. First, fibrin-Matrigel mixed gel was found to promote neural stem cell (NSC) differentiation into neurons and neurite extension. In particular, 3D neural networks were constructed in 2-8 mg/ml fibrin-Matrigel mixed gel. Second, we found that capillary-like structures were also formed in the fibrin-Matrigel mixed gel by coculturing brain microvascular endothelial cells (BMECs) and human mesenchymal stem cells (MSCs). Finally, we combined both neural and vascular culture models and succeeded in constructing 3D neurovascular tissues with an optimized seeding condition of NSCs, BMECs and MSCs.

Lysyl Oxidase Enhances the Deposition of Tropoelastin through the Catalysis of Tropoelastin Molecules on the Cell Surface.

The cross-linking of elastin by lysyl oxidase (LOX) family members is essential for the integrity and elasticity of elastic fibers, which play an important role in the characteristic resilience of various tissues. However, the temporal sequence of oxidation by LOX during elastic fiber formation is still incompletely understood. Here, we demonstrate that the cross-linking of tropoelastin molecules by LOX occurs concurrent with elastin deposition. Our data show that LOX deficiency or the inhibition of LOX enzyme activity leads to the loss of elastin deposition in skin fibroblast. Moreover, overexpression of LOX promotes the deposition and alignment of tropoelastin, whereas the addition of recombinant active-form of LOX in culture medium caused abnormal elastic fiber assembly. Immunoblotting and immunofluorescence show that LOX and tropoelastin are present together with fibronectin on the cell surface of preconfluent cultures. Further, fluorescence activated cell sorting (FACS) analysis for the localization of LOX on the cell surface reveals that the transfer of LOX to the extracellular space occurs in association with elastic fiber formation. In conclusion, our results support the view that LOX and tropoelastin are present on the cell surface and suggests the possibility that lysine oxidation by LOX precedes tropoelastin deposition onto microfibrils.

Endothelium-induced three-dimensional invasion of heterogeneous glioma initiating cells in a microfluidic coculture platform.

Glioblastoma (GBM) is a highly invasive primary brain tumor that displays cellular heterogeneity, which is composed of glioma initiating cells (GICs) and their differentiated progeny. GICs play an important role in driving aggressive invasion. In particular, the interaction between GICs and blood vessels is critical because blood vessels are known to serve as routes for the invasion of GICs. However, the effect of endothelial cells on the three-dimensional (3D) invasion process of GICs as well as the spatial relationship between GICs and their differentiated progeny remains unclear. Here, we utilized a microfluidic device to recapitulate the 3D brain tumor microenvironments constituted by human umbilical vein endothelial cells (HUVECs) and type I collagen. Using the device, we found that HUVECs promoted the 3D invasion of heterogeneous glioma cell populations into type I collagen gel. The invasion induced by HUVECs was predominantly preceded by cells positive for nestin, a neural stem cell marker. In contrast, cells positive for tubulin ß3 (TUBB3), a differentiated cell marker, rarely preceded invasion. In addition, HUVECs induced the upregulation of TUBB3 in GICs. Finally, we found that the genes associated with invasion, such as integrins α2 and ß3, were significantly upregulated in the presence of HUVECs. These results as well as the experimental approach provide valuable knowledge for the development of effective therapeutic strategies targeting the aggressive invasion of GBM.

A Novel Three-Dimensional Culture System for Oligodendrocyte Precursor Cells.

Oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs). Mechanisms of OPC differentiation have been extensively examined with two-dimensional cell culture systems. However, these cellular events may be more accurately represented using a three-dimensional (3D) model. In this study, we report the development of a novel 3D OPC culture system using gels composed of a mixture of collagen and hyaluronan, wherein cultured rat primary OPCs can proliferate and differentiate into oligodendrocytes. Our data show that the gel concentration and cell-seeding density are critical factors for the numbers of OPCs and oligodendrocytes in our 3D culture system. In addition, Notch signaling, which supports cell-to-cell communication, may also be important for OPC function in our system because a Notch inhibitor DAPT suppressed OPC proliferation and differentiation. Taken together, cultured rat OPCs can grow in collagen-/hyaluronan-based gels, and our novel 3D OPC culture system may offer a useful platform for examining the mechanisms of OPC function in vitro.

Fabrication of type I collagen microcarrier using a microfluidic 3D T-junction device and its application for the quantitative analysis of cell-ECM interactions.

We presented a new quantitative analysis for cell and extracellular matrix (ECM) interactions, using cell-coated ECM hydrogel microbeads (hydrobeads) made of type I collagen. The hydrobeads can carry cells as three-dimensional spheroidal forms with an ECM inside, facilitating a direct interaction between the cells and ECM. The cells on hydrobeads do not have a hypoxic core, which opens the possibility for using as a cell microcarrier for bottom-up tissue reconstitution. This technique can utilize various types of cells, even MDA-MB-231 cells, which have weak cell-cell interactions and do not form spheroids in conventional spheroid culture methods. Morphological indices of the cell-coated hydrobead visually present cell-ECM interactions in a quantitative manner.

MiR-29-mediated elastin down-regulation contributes to inorganic phosphorus-induced osteoblastic differentiation in vascular smooth muscle cells.

Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification.