Endothelial-mesenchymal Transformation Research Articles

Endothelial to Mesenchymal Transformation-derived Activated Fibroblast Behavior in a 3D Culture Environment

Objective: In calcific aortic valve disease (CAVD), glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and hyaluronic acid (HA) are present in the normally collagen-rich aortic valve fibrosa near calcified nodules. Previous work using a 3D in vitro model has shown that GAGs can also promote endothelial to mesenchymal transformation (EndMT). The role of mesenchymal transformation in the progression of CAVD remains unclear. The goal of this study is to investigate the role of GAGs and EndMT on valve cell behavior in a 3D, in vitro model of healthy and diseased valves. Methods: Three dimensional in-vitro cultures, representing healthy (collagen 1 hydrogels) or diseased (collagen 1 hydrogels + GAGs) valve tissue, were seeded with Porcine Aortic Valve Endothelial Cells (PAVEC) and Porcine Aortic Valve Interstitial Cells (PAVIC). Cell cultures were grown for 14 days in regular medium and then evaluated for calcified nodule formation, alkaline phosphatase activity, proliferation and protein expression relating to EndMT activity Results: Introduction of GAGs into the in vitro model resulted in decreased alkaline phosphatase activity. Hydrogels containing CS conditions induced the greatest calcification, as measured with Alzarin Red quantification, while gels containing HA conditions did not have calcification that was significantly different than controls. Proliferation results suggest high levels of cell division activity, for both PAVEC and PAVIC in CS conditions. Protein expression analysis indicated increased EndMT in hydrogels containing GAGs. Conclusions: 3D, in vitro models of the aortic valve simulated progression toward late stage CAVD. Further investigation of the downstream role of EndMT within the aortic valve microenvironment is critical to subsequent improvement in treatment options.

The role of β-catenin in pulmonary artery endothelial-mesenchymal transformation in rats with chronic thromboembolic pulmonary hypertension.

β-catenin and endothelial mesenchymal transformation play an important role in the formation of pulmonary hypertension. To explore the role of β-catenin in chronic thromboembolic pulmonary hypertension (CTEPH), we first established a rat model of CTEPH by repeated autologous thromboembolization and then treated these rats with a β-catenin specific inhibitor, XAV939, for two or four weeks. We further examined the expression of β-catenin, α-SMA and CD31, mean pulmonary artery pressure (mPAP), and histopathology in the pulmonary artery, and analyzed their correlation. In the thrombus group without treatment of the inhibitor, the expression of β-catenin and α-SMA in pulmonary artery was increased with time; mPAP, the thickness of pulmonary artery wall, and the area/total area of pulmonary artery (WA/TA) were also increased; however, the expression of CD31 was decreased. Interestingly, these symptoms could be improved by treatment with XAV939. In this study, in CTEPH rat model, the expression of β-catenin signal affects pulmonary vascular remodeling and pulmonary artery pressure, and positively correlated with pulmonary arterial endothelial mesenchymal transformation (EMT), indicating that β-catenin signal may play an important role in the occurrence and development of CTEPH. The inhibition of β-catenin signal and the improvement of pulmonary arterial EMT may provide therapeutic ideas for CTEPH.

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Low shear stress-induced endothelial mesenchymal transformation via the down-regulation of TET2

Atherosclerotic cardiovascular disease is the major cause of death worldwide. Low shear stress plays key roles on the initiation and progression of atherosclerosis (As). However, its underlying mechanism remains unclear. In this study, the effect of low shear stress on endothelial mesenchymal transformation (EndMT) and its underlying mechanism were explored. Results showed that in cultured human umbilical vein endothelial cells, low shear stress down-regulated the expression of TET2 and promoted EndMT. Loss of TET2 promoted EndMT with the Wnt/β-catenin signaling pathway. The enhancement in EndMT induced by low shear stress was attenuated by TET2 overexpression. In apoE−/− mice subjected to carotid artery local ligation, the EndMT and atherosclerotic lesions induced by low shear stress was attenuated by TET2 overexpression. Taken together, low shear stress promoted EndMT through the down-regulation of TET2, indicating that intervention with EndMT or the up-regulation of TET2 might be an alternative strategy for preventing As.

Valve endothelial-interstitial interactions drive emergent complex calcific lesion formation in vitro

ObjectiveCalcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) – interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments. Approach and resultsPorcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11–7082. ConclusionsOur results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control.

MiR-217 inhibits apoptosis of atherosclerotic endothelial cells via the TLR4/PI3K/Akt/NF-κB pathway.

To determine the effect of miR-217 on the apoptosis of atherosclerotic endothelial cells (AECs) through the Toll-like receptor (TLR) 4/PI3K/Akt/NF-κB pathway. Oxidized low-density lipoprotein (ox-LDL) was used to construct an atherosclerotic endothelial cell model, and the expression of miR-217/TLR4/PI3K/Akt/NF-κB in the cells was regulated to explore their effects on the viability, apoptosis, inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10)], and endothelial-to-mesenchymal transformation (EndMT) of the endothelial cells. In AECs, miR-217 expression decreased, and the PI3K/Akt/NF-κB pathway was inhibited. The Dual-Luciferase reporter assay revealed that TLR4 was the target of miR-217, and it was up-regulated in AECs, and the further study revealed that up-regulation of miR-217 protected AECs, increased their activity, reduced their apoptosis, and inhibited inflammatory response and EndMT, while TLR4 acted contrary to miR-217. Besides, it was also found that miR-217 inhibited the PI3K/Akt/NF-κB pathway, thus weakening the influence of si-TLR4 on endothelial cells. Furthermore, miR-217 inhibited EndMT by inhibiting TLR4 from activating the PI3K/Akt/NF-κB signal pathway. In AECs, TLR4 expression increased, and miR-217 and the PI3K/Akt/NF-κB signaling pathway are inhibited. Additionally, miR-217 can increase the viability of AECs through the TLR4/PI3K/Akt/ NF-κB signal transduction pathway, and inhibit their apoptosis, inflammatory response, and EndMT.

Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-\u03baB axis dependent manner

Cardiovascular disease is still the leading cause of mortality worldwide. Vascular endothelial dysfunction is viewed as the initial step of most cardiovascular diseases. Many studies have indicated that periodontal pathogens, especially Porphyromonas gingivalis, are closely correlated with vascular endothelial homeostasis, but the function of P. gingivalis and the underlying mechanisms are still elusive. To illuminate the effects and elucidate the mechanisms of P. gingivalis on endothelial structural integrity, we developed P. gingivalis infection models in vivo and in vitro. Endothelial cell proliferation, differentiation and apoptosis were detected. Here, we showed that P. gingivalis can impair endothelial integrity by inhibiting cell proliferation and inducing endothelial mesenchymal transformation and apoptosis of endothelial cells, which reduce the cell levels and cause the endothelium to lose its ability to repair itself. A mechanistic analysis showed that TLR antagonist or NF-κB signalling inhibitor can largely rescue the damaged integrity of the endothelium caused by P. gingivalis, suggesting that TLR-NF-κB signalling plays a vital role in vascular endothelial homeostasis destroyed by P. gingivalis. These results suggest a potential intervention method for the prevention and treatment of cardiovascular disease.

Side-dependent effect in the response of valve endothelial cells to bidirectional shear stress

Endothelial cells covering the aortic and ventricular sides of the aortic valve leaflets are exposed to different stresses, in particular wall shear stress (WSS). Biomechanical stimuli actively regulate valve tissue structure and induce remodeling events leading to valve dysfunction. Endothelial to mesenchymal transformation (EndMT), for example, has been associated with aortic valve disease. The biomechanical response of cells at different sides of the leaflets has not been clearly characterized. To analyze the mechanical response of valve endothelial cells (VECs) we developed a unique fluid activation device that applies physiologically relevant pulsatile WSS. We characterized the morphology and function of adult porcine aortic VECs derived from the opposite sides of aortic valve leaflets following exposure to different pulsatile WSS. We found that elongation and orientation of cells in response to pulsatile WSS depends on their side of origin. Quantification of gene expression confirms phenotypic differences between aortic and ventricular VECs. Aortic VECs exposed to pulsatile WSS similar to that in vivo at the tip of aortic side of the valve leaflet upregulated pro-EndMT (ACTA2, Snail, TGFβ1) and inflammation (ICAM-1, VCAM-1) genes, whereas expression of endothelial markers like PECAM-1 was decreased. Conversely, ventricular-VECs showed strong increase of PECAM-1 expression and no activation of pro-EndMT marker. Finally, we found that stress-induced genes are upregulated in both cell types, at higher levels in ventricular compared to aortic VECs. Application of physiological shear stress levels using a fluid activation device therefore reveals functional differences in VECs derived from opposite sides of the aortic valve leaflets.

NLRC5 deficiency ameliorates cardiac fibrosis in diabetic cardiomyopathy by regulating EndMT through Smad2/3 signaling pathway

Diabetic cardiomyopathy (DCM) is one of the main causes of heart failure in patients with diabetes. Cardiac fibrosis caused by endothelial mesenchymal transformation (EndMT) plays an important role in the pathogenesis of DCM. NLRC5 is a recently discovered immune and inflammatory regulatory molecule in the NOD-like receptor family, and is involved in organ fibrosis. In this study, we found that the expression of NLRC5 was up-regulated in endothelial cells (ECs) and cardiac fibroblasts (CFs) in diabetes models both in vivo and in vitro. NLRC5 knockdown significantly inhibited high glucose-induced EndMT. In addition, NLRC5 deficiency inhibited the expression of phosphorylated Smad2/3 and the activation of EndMT-related transcription factors in ECs induced by high glucose. However, the effect of NLRC5 deficiency on CFs was not obvious. In summary, our results suggest that NLRC5 deficiency ameliorates cardiac fibrosis in DCM by inhibiting EndMT through Smad2/3 signaling pathway and related transcription factors. NLRC5 is likely to be a biomarker and therapeutic target of cardiac fibrosis in diabetic cardiomyopathy.

CircRNA_100367 regulated the radiation sensitivity of esophageal squamous cell carcinomas through miR-217/Wnt3 pathway.

Background: Circular RNAs (circRNAs) play important roles in regulating the radioresistance of esophageal squamous cell carcinoma (ESCC). This study aimed to determine the role of hsa_circRNA_100367 in regulating radioresistance of ESCC.Results: Higher expression and potency of endothelial to mesenchymal transformation (EMT) was found in radioresistant ESCC cells (KYSE-150R) than in ESCC cells (KYSE-150). Silencing circRNA_100367 inhibited the proliferation and migration of KYSE-150R cells, and decreased the expression of β-catenin (an important molecule in Wnt pathway) in KYSE-150R cells. Additionally, circRNA_100367 bound to miR-217, and miR-217 targeted Wnt3. Low Wnt3 expression was associated with the short survival time in patients with ESCC and Wnt3 knockdown inhibited the proliferation and migration of KYSE-150R cells. CircRNA_100367 enhanced the radioresistance of KYSE-150R cells through miR-217/Wnt3 pathway. In vivo, circRNA_100367 silence reduced the growth of KYSE-150R cells under radiation.Conclusion: Our results revealed that circRNA_100367 attenuated radioresistance of ESCC through miR-217/Wnt3 pathway.Methods: CircRNAs related with the radioresistance of ESCC were analyzed by hierarchical cluster analysis. The relationship between circRNA_100367 and miR-217, Wnt3 was detected by RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporte assays. The proliferation and migration ESCC cells were detected by MTT, Transwell and colony formation assays.

A multiscale in silico model of endothelial to mesenchymal transformation in a tumor microenvironment

Endothelial to mesenchymal transformation (EndMT) is a process in which endothelial cells gain a mesenchymal-like phenotype in response to mechanobiological signals that results in the remodeling or repair of underlying tissue. While initially associated with embryonic development, this process has since been shown to occur in adult tissue remodeling including wound healing, fibrosis, and cancer. In an attempt to understand the role of EndMT in cancer progression and metastasis, we present a multiscale, three-dimensional, in silico model. The model couples tissue level phenomena such as extracellular matrix remodeling, cellular level phenomena such as migration and proliferation, and chemical transport in the tumor microenvironment to mimic in vitro tissue models of the cancer microenvironment. The model is used to study the presence of EndMT-derived activated fibroblasts (EDAFs) and varying substrate stiffness on tumor cell migration and proliferation. The simulations accurately model the behavior of tumor cells under given conditions. The presence of EDAFs and/or an increase in substrate stiffness resulted in an increase in tumor cell activity. This model lays the foundation of further studies of EDAFs in a tumor microenvironment on a cellular and subcellular physiological level.

YAP-Induced Endothelial-Mesenchymal Transition in Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a potentially malignant disorder. Current studies have shown that chewing areca nut is considered the main cause of OSF, and endothelial-mesenchymal transformation (EndMT) participates in the occurrence and development of the fibrotic lesion. However, the specific molecular mechanisms and treatments remain unclear. Here, we report the mechanism of arecoline-induced EndMT and the importance of this mechanism in OSF, and we also identify potential therapeutics for decreasing OSF incidence. We demonstrate the overexpression of Yes-associated protein (YAP) in human samples and that it was significantly associated with OSF pathologic stage. Arecoline activated YAP by increasing reactive oxygen species levels and inducing the PERK pathway (eukaryotic translation initiation factor 2 alpha kinase 3), resulting in the initiation of EndMT and leading to OSF. Verteporfin, a YAP–TEA domain pathway inhibitor, suppressed EndMT and decreased collagen accumulation, resulting in the alleviation of OSF in mice. These data indicate that arecoline regulates the activity of YAP and highlight an alternative method for treating OSF.

A Rabbit Corneal Endothelial Dysfunction Model Using Endothelial-Mesenchymal Transformed Cells

Unlike humans, rabbit corneal endothelial wounds are known to spontaneously heal. The current study was aimed to develop a new rabbit bullous keratopathy model using corneal endothelial cells that were induced to undergo endothelial-mesenchymal transformation (EMT). EMT was induced in rabbit corneal endothelial cells (RCECs) by culturing with TGFβ and basic FGF Supplemented Medium. The corneal endothelia in recipient rabbits were mechanically scraped from the corneal endothelial surface inside an 8 mm mark. Then, a suspension of EMT-induced RCECs (EMT-RCECs) was injected into the anterior chamber. Eyes injected with freshly isolated RCECs (Fresh RCECs group) and eyes that were scraped without injection of cells (Scrape group) were used as controls. Immediately following operation, subepithelial and stromal edema was observed with increased central corneal thickness and corneal opacity in all groups. In the EMT-RCECs group, bullous keratopathy persisted for 42 days up to the end of the study. In the Fresh-RCECs and Scrape groups, corneal transparency and thickness recovered by 7 days after treatment and was maintained up to 42 days. The activated fibroblast marker, α-SMA, was observed spanning from corneal endothelium to corneal stroma in the EMT-RCECs group. Interestingly, α-SMA was upregulated in the Scrape-group as well. In all groups, there was no damage to other intraocular structures, and intraocular pressure was normal throughout the observation period. Transplanting a fresh donor cornea effectively treated corneal edema due to bullous keratopathy. This model is a promising tool for pre-clinical trials in the development of new therapies against corneal endothelial dysfunction.

PDGF-mediated mesenchymal transformation renders endothelial resistance to anti-VEGF treatment in glioblastoma

Angiogenesis is a hallmark of cancer. However, most malignant solid tumors exhibit robust resistance to current anti-angiogenic therapies that primarily target VEGF pathways. Here we report that endothelial-mesenchymal transformation induces glioblastoma (GBM) resistance to anti-angiogenic therapy by downregulating VEGFR-2 expression in tumor-associated endothelial cells (ECs). We show that VEGFR-2 expression is markedly reduced in human and mouse GBM ECs. Transcriptome analysis verifies reduced VEGFR-2 expression in ECs under GBM conditions and shows increased mesenchymal gene expression in these cells. Furthermore, we identify a PDGF/NF-κB/Snail axis that induces mesenchymal transformation and reduces VEGFR-2 expression in ECs. Finally, dual inhibition of VEGFR and PDGFR eliminates tumor-associated ECs and improves animal survival in GBM-bearing mice. Notably, EC-specific knockout of PDGFR-β sensitizes tumors to VEGF-neutralizing treatment. These findings reveal an endothelial plasticity-mediated mechanism that controls anti-angiogenic therapy resistance, and suggest that vascular de-transformation may offer promising opportunities for anti-vascular therapy in cancer.

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells.

LY2109761, Transforming Growth Factor β Receptor Type I and Type II Dual Inhibitor, is a Novel Approach to Suppress Endothelial Mesenchymal Transformation in Human Corneal Endothelial Cells

Background/Aims: Preventing undesirable endothelial-mesenchymal transformation (EnMT) with repetitious in vitro expansion of human corneal endothelial cells (CECs) is a pivotal issue in cornea regeneration. Previous studies have shown that inhibition of the TGF-β pathway reduces epithelial-mesenchymal transformation. However, its potential role in EnMT remains poorly understood. As such, the effect of LY2109761, a novel TGF-β receptor type I and type II dual inhibitor, was investigated on EnMT. Methods: CECs cultured with various concentrations of LY2109761 were evaluated for their growth rate and phenotype. Additionally, the expression of functional markers (sodium-potassium pump Na⁺/K⁺-ATPase and the tight junction protein ZO-1) and mesenchymal markers (CD73, fibronectin, and vimentin) was detected using immunostaining and western blot. The mRNA expressions were also assayed by real-time polymerase chain reaction analysis. Results: At a 1 μM concentration, LY2109761 did not influence the proliferation of CECs and subsequent experiments were therefore performed using this concentration. Furthermore, CECs cultured in the presence of 1 μM LY2109761 maintained their ability to grow as a monolayer of hexagonal-shaped cells. The expression of functional markers increased in LY2109761-treated CECs, while the expression of mesenchymal markers decreased (both in protein and mRNA levels). Conclusion: Inhibition of TGF-β receptor type I and type II by LY2109761 maintained the phenotype of CECs and inhibited the EnMT process. These results indicate the possible continuous in vitro expansion of CECs with normal function.

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm2) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate, distance, and proliferation are induced by EndMT-derived activated fibroblasts. This model can be used to explore new therapeutics in a tumor microenvironment.

Endothelial to mesenchymal transformation is induced by altered extracellular matrix in aortic valve endothelial cells

Alterations in shear stress, mechanical deformation, extracellular matrix (ECM) composition and exposure to inflammatory conditions are known to cause endothelial to mesenchymal transformation (EndMT). This change in endothelial phenotype has only recently been linked to adult pathologies such as cancer progression, organ fibrosis, and calcific aortic valve disease; and its function in adult physiology, especially in response to tissue mechanics, has not been rigorously investigated. EndMT is a response to mechanical and biochemical signals that results in the remodeling of underlying tissues. In diseased aortic valves, glycosaminoglycans (GAGs) are present in the collagen-rich valve fibrosa, and are deposited near calcified nodules. In this study, in vitro models of early and late-stage valve disease were developed by incorporating the GAGs chondroitin sulfate (CS), hyaluronic acid, and dermatan sulfate into 3D collagen hydrogels with or without exposure to TGF-β1 to simulate EndMT in response to microenvironmental changes. High levels of CS induced the highest rate of EndMT and led to the most collagen I and GAG production by mesenchymally transformed cells, which indicates a cell phenotype most likely to promote fibrotic disease. Mesenchymal transformation due to altered ECM was found to depend on cell-ECM bond strength and extracellular signal-regulated protein kinases 1/2 signaling. Determining the environmental conditions that induce and promote EndMT, and the subsequent behavior of mesenchymally transformed cells, will advance understanding on the role of endothelial cells in tissue regeneration or disease progression. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2729-2741, 2017.

In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers

Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers in vitro.

ANGI-01. TRANSFORMATION OF TUMOR ENDOTHELIAL CELLS TO MESENCHYMAL STEM CELL-LIKE CELLS INDUCES THERAPY RESISTANCE IN GLIOBLASTOMA

Newly formed tumor blood vessels deliver oxygen and nutrients and produce paracrine factors to tumor microenvironment, fueling tumor growth, progression and metastasis. Likewise, targeting vascular endothelial cells (ECs) has emerged as a fundamental strategy for cancer treatment. However, inefficient eradication and insufficient functional inhibition of tumor-associated ECs remains a major barrier for current anti-vascular therapies. As such, current anti-angiogenic therapies that mainly target vascular endothelial growth factor (VEGF) have encountered difficulties and failure in most types of malignant cancers. Our recent work has identified endothelial mesenchymal transformation in glioblastoma (GBM), which leads to the suppression of VEGF signaling in GBM-associated ECs and renders intrinsic EC resistance to anti-VEGF treatment (Huang et al, JCI 2016). Here, we reveal cell plasticity in ECs drives their resistance to chemotherapy. Our data show that tumor ECs derived from human GBM patients are resistant to chemotherapy, and the resistance correlates with the mesenchymalization in ECs. Utilizing EC lineage tracing in a genetic murine GBM model, we show that mesenchymal transformation induces EC resistance to temozolomide (TMZ) chemotherapy. Interestingly, transcriptome analysis by RNA deep sequencing reveals that ECs undergo transformation to mesenchymal stem cell (MSC)-like cells after expose to GBM microenvironment. ECs acquire MSC signature genes and stemness-associated transcriptional activation, leading to chemoresistance. Our further mechanistic study identifies a c-Met-mediated axis that induces canonical activation of Wnt signaling. Finally, genetic ablation and pharmacological inhibition of c-Met reduce EC transformation and overcome chemoresistance in ECs. These findings illustrate a cell transformation-based mechanism that controls chemoresistance in tumor ECs, and suggest that targeting c-Met may sensitize ECs to chemotherapy and improve anti-vascular treatment for cancers.