CD133-Src-TAZ signaling stimulates ductal fibrosis following DDC diet-induced liver injury.

Chronic liver injury follows inflammation and liver fibrosis; however, the molecular mechanism underlying fibrosis has not been fully elucidated. In this study, the role of ductal WW domain-containing transcription regulator 1 (WWTR1)/transcriptional coactivator with PDZ-binding motif (TAZ) was investigated after liver injury. Ductal TAZ-knockout (DKO) mice showed decreased liver fibrosis following a Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) diet compared to wild-type (WT) mice, as evidenced by decreased expression levels of fibrosis inducers, including connective tissue growth factor (Ctgf)/cellular communication network factor 2 (CCN2), cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), and transforming growth factor beta 1 (Tgfb1), in DKO mice. Similarly, TAZ-knockout (KO) cholangiocyte organoids showed decreased expression of fibrosis inducers. Additionally, the culture supernatant of TAZ-KO cholangiocyte organoids decreased the fibrogenic gene expression in liver stellate cells. Further studies revealed that prominin 1 (PROM1/CD133) stimulated TAZ for fibrosis. After the administration of DDC diet, fibrosis was decreased in CD133-KO (CD133-KO) mice compared to that in WT mice. Similarly, CD133-KO cholangiocyte organoids showed decreased Ctgf, Cyr61, and Tgfb1 expression levels compared to WT cholangiocyte organoids. Mechanistically, CD133 stabilized TAZ via Src activation. Inhibition of Src decreased TAZ levels. Similarly, CD133-knockdown HCT116 cells showed decreased TAZ levels, but reintroduction of active Src recovered the TAZ levels. Taken together, our results suggest that TAZ facilitates liver fibrosis after a DDC diet via the CD133-Src-TAZ axis.

TAZ links exercise to mitochondrial biogenesis via mitochondrial transcription factor A.

Mitochondria are energy-generating organelles and mitochondrial biogenesis is stimulated to meet energy requirements in response to extracellular stimuli, including exercise. However, the mechanisms underlying mitochondrial biogenesis remain unknown. Here, we demonstrate that transcriptional coactivator with PDZ-binding motif (TAZ) stimulates mitochondrial biogenesis in skeletal muscle. In muscle-specific TAZ-knockout (mKO) mice, mitochondrial biogenesis, respiratory metabolism, and exercise ability were decreased compared to wild-type mice. Mechanistically, TAZ stimulates the translation of mitochondrial transcription factor A via Ras homolog enriched in brain (Rheb)/Rheb like 1 (Rhebl1)-mTOR axis. TAZ stimulates Rhebl1 expression via TEA domain family transcription factor. Rhebl1 introduction by adeno-associated virus or mTOR activation recovered mitochondrial biogenesis in mKO muscle. Physiologically, mKO mice did not stimulate exercise-induced mitochondrial biogenesis. Collectively, our results suggested that TAZ is a novel stimulator for mitochondrial biogenesis and exercise-induced muscle adaptation.

Stochastic chromatin packing of 3D mitotic chromosomes revealed by coherent X-rays.

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.

Transcriptional coactivator with PDZ-binding motif stimulates epidermal regeneration via induction of amphiregulin expression after ultraviolet damage.

Ultraviolet (UV) irradiation induces the proliferation and differentiation of keratinocytes in the basal layer of the epidermis, which increases epidermal thickness in skin regeneration. However, the mechanism underlying this phenomenon is not yet known in detail. In this study, we aimed to demonstrate that the transcriptional coactivator with PDZ-binding motif (TAZ) stimulates epidermal regeneration by increasing keratinocyte proliferation. During epidermal regeneration, TAZ is localized in the nucleus of keratinocytes of the basal layer and stimulates epidermal growth factor receptor (EGFR) signaling. TAZ depletion in keratinocytes decreased EGFR signaling activation, which delays epidermal regeneration. Interestingly, TAZ stimulated the transcription of amphiregulin (AREG), a ligand of EGFR, through TEAD-mediated transcriptional activation. Together, these results show that TAZ stimulates EGFR signaling through AREG induction, suggesting that it plays an important role in epidermal regeneration.

Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell.

A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.

TAZ stimulates liver regeneration through interleukin-6-induced hepatocyte proliferation and inhibition of cell death after liver injury.

In response to liver injury, the liver undergoes a regeneration process to retain its mass and function. However, the regeneration mechanism has not been fully clarified. This study investigated the role of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo-signaling effector, in liver regeneration. We observed that TAZ stimulates liver regeneration after liver injury. After partial hepatectomy (PHx) or carbon tetrachloride damage, TAZ was required for liver regeneration to increase hepatic cell proliferation and resist hepatic apoptosis, which were decreased in liver-specific TAZ knockout (LKO) mice. TAZ stimulated macrophage infiltration, resulting in IL-6 production, which induced liver regeneration. In LKO mice, IL-6-induced activation of signal transducer and activator of transcription 3, ERK, and PKB was decreased. We also observed that periductal fibrogenesis was significantly increased in LKO mice during liver regeneration after PHx, which was caused by increased hepatic apoptosis. Our results suggest that TAZ stimulates liver regeneration through IL-6-induced hepatocyte proliferation and inhibition of cell death after liver injury.-Kim, A. R., Park, J. I., Oh, H. T., Kim, K. M., Hwang, J.-H., Jeong, M. G., Kim, E.-H., Hwang, E. S., Hong, J.-H. TAZ stimulates liver regeneration through interleukin-6-induced hepatocyte proliferation and inhibition of cell death after liver injury.

Artificial cellular nano-environment composed of collagen-based nanofilm promotes osteogenic differentiation of mesenchymal stem cells.

In regenerative medicine, the generation of therapeutic stem cells and tissue engineering are important for replacing damaged tissues. Numerous studies have attempted to produce cellular components that mimic the native tissue for gaining optimal function. Particularly, the extracellular matrix (ECM) composition plays an important role in cellular functions including determining the fates of mesenchymal stem cells (MSCs). Here, we evaluated the osteogenic effects of a nanofilm in which oppositely charged polyelectrolytes were alternately adsorbed onto the cell surface to create an artificial ECM environment for single MSCs. Interestingly, nanofilm composed of collagen (Col) and alginate (AA) showed relatively high stiffness and MSCs coated with the Col/AA nanofilm showed increased osteogenic differentiation efficiency compared to other nanofilm-coated MSCs. Further analysis revealed that the Col/AA nanofilm coating stimulated osteogenesis by activating transcriptional coactivators with the PDZ binding motif through extracellular signal-related kinase and p38 MAPK signaling. This nano-sized cellular coating will facilitate the development of nanotechnology for controlling cellular functions and advance stem cell-based clinical applications for regenerative medicine. STATE OF SIGNIFICANCE: In this study, we developed an artificial cellular nano-environment formed by multilayer nanofilms. We demonstrated that the nanofilms introduced to mesenchymal stem cells (MSCs) stimulate osteogenic differentiation by regulating intracellular signaling. Among the various nanofilm combinations, the induction of osteogenic gene transcription in collagen (Col) and alginate (AA) film-coated MSCs was the most pronounced compared to that on other nanofilms. A minimum number of Col/AA nanofilm bilayers (n = 2) was required for effective induction of MSC osteogenic differentiation. In addition, we observed the correlation between the promoting effect of osteogenic differentiation and stiffness of the nanofilm. Our results may be useful for developing a cell coating model system widely applicable in bioengineering and regenerative medicine.

SRC activates TAZ for intestinal tumorigenesis and regeneration.

Proto-oncogene tyrosine-protein kinase Src (cSRC) is involved in colorectal cancer (CRC) development and damage-induced intestinal regeneration, although the cellular mechanisms involved are poorly understood. Here, we report that transcriptional coactivator with PDZ binding domain (TAZ) is activated by cSRC, regulating CRC cell proliferation and tumor formation, where cSRC overexpression increases TAZ expression in CRC cells. In contrast, knockdown of cSRC decreases TAZ expression. Additionally, direct phosphorylation of TAZ at Tyr316 by cSRC stimulates nuclear localization and facilitates transcriptional enhancer factor TEF-3 (TEAD4)-mediated transcription. However, a TAZ phosphorylation mutant significantly decreased cell proliferation, wound healing, colony forming, and tumor formation. In a CRC mouse model, ApcMin/+, activated SRC expression was associated with increased TAZ expression in polyps and TAZ depletion decreased polyp formation. Moreover, intestinal TAZ knockout mice had intestinal regeneration defects following γ-irradiation. Finally, significant correspondence between SRC activation and TAZ overexpression was observed in CRC patients. These results suggest that TAZ is a critical factor for SRC-mediated intestinal tumor formation and regeneration.

Nanotopological plate stimulates osteogenic differentiation through TAZ activation.

The topographical environment, which mimics the stem cell niche, provides mechanical cues to regulate the differentiation of mesenchymal stem cells (MSC). Diverse topographical variations have been engineered to investigate cellular responses; however, the types of mechanical parameters that affect cells, and their underlying mechanisms remain largely unknown. In this study, we screened nanotopological pillars with size gradient to activate transcriptional coactivator with PDZ binding motif (TAZ), which stimulates osteogenesis of MSC. We observed that a nanotopological plate, 70 nm in diameter, significantly induces osteogenic differentiation with the activation of TAZ. TAZ activation via the nanotopological plate was mediated by actin polymerization and Rho signaling, as evidenced by the cytosolic localization of TAZ under F-actin or Rho kinase inhibitor. The FAK and MAPK pathways also play a role in TAZ activation by the nanotopological plate because the inhibitor of ERK and JNK blocked nanopattern plate induced osteogenic differentiation. Taken together, these results indicate that nanotopology regulates cell differentiation through TAZ activation.

Catechins activate muscle stem cells by Myf5 induction and stimulate muscle regeneration.

Muscle weakness is one of the most common symptoms in aged individuals and increases risk of mortality. Thus, maintenance of muscle mass is important for inhibiting aging. In this study, we investigated the effect of catechins, polyphenol compounds in green tea, on muscle regeneration. We found that (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) activate satellite cells by induction of Myf5 transcription factors. For satellite cell activation, Akt kinase was significantly induced after ECG treatment and ECG-induced satellite cell activation was blocked in the presence of Akt inhibitor. ECG also promotes myogenic differentiation through the induction of myogenic markers, including Myogenin and Muscle creatine kinase (MCK), in satellite and C2C12 myoblast cells. Finally, EGCG administration to mice significantly increased muscle fiber size for regeneration. Taken together, the results suggest that catechins stimulate muscle stem cell activation and differentiation for muscle regeneration.

(-)-Epigallocatechin-3-gallate stimulates myogenic differentiation through TAZ activation.

Muscle loss is a typical process of aging. Green tea consumption is known to slow down the progress of aging. Their underlying mechanisms, however, remain largely unknown. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, on myogenic differentiation and found that EGCG significantly increases myogenic differentiation. After EGCG treatment, the expression of myogenic marker genes, such as myosin heavy chain, are increased through activation of TAZ, a transcriptional coactivator with a PDZ-binding motif. TAZ-knockdown does not stimulate EGCG-induced myogenic differentiation. EGCG facilitates the interaction between TAZ and MyoD, which stimulates MyoD-mediated gene transcription. EGCG induces nuclear localization of TAZ through the dephosphorylation of TAZ at its Ser89 residue, which relieves 14-3-3 binding in the cytosol. Interestingly, inactivation of Lats kinase is observed after EGCG treatment, which is responsible for the production of dephosphorylated TAZ. Together, these results suggest that EGCG induces myogenic differentiation through TAZ, suggesting that TAZ plays an important role in EGCG induced muscle regeneration.

Tunable Crosslinked Cell-Derived Extracellular Matrix Guides Cell Fate.

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells.

BACKGROUND: The vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) signaling pathway is involved in cancer-related biological functions and is a therapeutic target in cancer. However, the influence of epigenetic regulation of VEGF-VEGFR signaling-related genes remains unclear. Here, we evaluated the effects of FLT1 and KDR promoter hypermethylation combined with drugs targeting VEGF-VEGFR signaling on cancer-related phenotypes in renal cancer cells (RCCs) and examined changes in FLT1 and KDR promoter hypermethylation in tissues from patients with renal cancer. RESULTS: In vitro experiments were performed to evaluate the effects of beavacizumab (an anti-VEGF antibody), an anti-FLT1 peptide, an anti-KDR antibody, and the VEGFR tyrosine kinase inhibitors (TKIs) sunitinib and axitinib in 13 RCC lines with different levels of FLT1 and/or KDR promoter methylation and in 2 FLT1 or KDR in vitro knockdown models. The synergistic effects of sunitinib and axitinib treatment were also evaluated in four RCC lines having different levels of FLT1 and/or KDR methylation. In our in vitro experiments, bevacizumab and an anti-KDR antibody did not affect the proliferation of RCCs having FLT1 and/or KDR hypermethylation. In contrast, in RCCs with FLT1 hypermethylation, proliferation inhibition was counteracted by treatment with an anti-FLT1 peptide and both VEGF-TKIs (sunitinib and axitinib). Demethylation with sunitinib or axitinib synergistically increased proliferation inhibition in the RCCs exhibiting FLT1 hypermethylation. Using in vitro FLT1 or KDR knockdown models, decreased proliferation inhibition following anti-FLT1 peptide, sunitinib, and axitinib treatment was observed only in FLT1-knockdown cells. In patients with renal cancer who received sunitinib, FLT1 promoter methylation was higher in renal cancer tissues from eight nonresponders (stable or progressive disease assessed by the Response Evaluation Criteria in Solid Tumors) than in cancer tissues from five responders (complete response or partial response). CONCLUSIONS: The present data showed that hypermethylated FLT1 was important for the efficacy of anti-VEGF/VEGFR drugs targeting FLT1 or intracellular VEGFR signaling. FLT1 hypermethylation causing alterations of FLT1 function could serve as a useful biomarker for predicting changes in FLT1 status in RCCs.

Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC) differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

FGF2 stimulates osteogenic differentiation through ERK induced TAZ expression.

TAZ (transcriptional coactivator with PDZ-binding motif) is a transcriptional modulator that regulates mesenchymal stem cell differentiation. It stimulates osteogenic differentiation while inhibiting adipocyte differentiation. FGFs (fibroblast growth factors) stimulate several signaling proteins to regulate their target genes, which are involved in cell proliferation, differentiation, and cell survival. Within this family, FGF2 stimulates osteoblast differentiation though a mechanism that is largely unknown. In this report, we show that TAZ mediates FGF2 signaling in osteogenesis. We observed that FGF2 increases TAZ expression by stimulating its mRNA expression. Depletion of TAZ using small hairpin RNA blocked FGF2-mediated osteogenic differentiation. FGF2 induced TAZ expression was stimulated by ERK (extracellular signal-regulated kinase) activation and the inhibition of ERK blocked TAZ expression. FGF2 increased nuclear localization of TAZ and, thus, facilitated the interaction of TAZ and Runx2, activating Runx2-mediated gene transcription. Taken together, these results suggest that TAZ is an important mediator of FGF2 signaling in osteoblast differentiation.

Idesolide inhibits the adipogenic differentiation of mesenchymal cells through the suppression of nitric oxide production.

Obesity is a major health problem worldwide and can increase the risk for several chronic diseases, including diabetes and cardiovascular disease. In this study, we screened small compounds isolated from natural products for the development of an anti-obesity drug. Among them, idesolide, a spiro compound isolated from the fruits of Idesia polycarpa Maxim, showed a significant suppression of the adipogenic differentiation in mesenchymal cells, as indicated by the decrease in fat droplets and expression of adipogenic marker genes such as aP2 and adiponectin. Idesolide inhibits the PPARγ-mediated gene transcription in a dose-dependent manner, revealed by luciferase reporter gene assay. During adipogenic differentiation, idesolide inhibits nitric oxide production through the suppression of iNOS expression, and the increased adipogenic differentiation by arginine, the substrate for NOS, is significantly inhibited by idesolide, suggesting that the inhibition of nitric oxide production plays a major role in idesolide-induced adipogenic suppression. Taken together, the results reveal that idesolide has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity.

Phorbaketal A stimulates osteoblast differentiation through TAZ mediated Runx2 activation.

Osteoporosis arises from an imbalance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we screened molecules from marine natural products that stimulate osteoblast differentiation. We found that phorbaketal A significantly stimulates osteoblast differentiation in mesenchymal cells. Increased interaction of TAZ and Runx2 stimulated phorbaketal A-induced expression of osteoblastic marker genes. The activation of ERK was important for the stimulation of differentiation because an inhibitor of ERK blocked phorbaketal A-induced osteogenic differentiation. Taken together, the results showed that phorbaketal A stimulates TAZ-mediated osteoblast differentiation through the activation of ERK.

Promoter methylation status of VEGF receptor genes: a possible epigenetic biomarker to anticipate the efficacy of intracellular-acting VEGF-targeted drugs in cancer cells.

We evaluated whether the inhibitory effects of vascular endothelial growth factor (VEGF)-targeted drugs on the proliferation of cancer cells differed according to VEGF receptor (VEGFR) genes, Flt1 and KDR, promoter methylation status. Five hyper-VEGFR-methylation and six no-VEGFR-methylation cancer cells were used for the present study, together with human umbilical endothelial cells (HUVECs) as a control. No-VEGFR-methylation cancer cells showed higher expression of Flt1 and KDR than hyper-VEGFR-methylation cancer cells. Hyper-VEGFR-methylation cancer cells only showed increased expression and protein levels of Flt1 and KDR after treatment with the demethylase 5-aza-2'-deoxycytidine. Two drugs (a VEGF-specific-antibody, bevacizumab, and a KDR-specific-antibody) targeting extracellular VEGF-VEGFR signaling and two VEGF-specific-tyrosine kinase inhibitors (PTK/ZK and sunitinib) targeting intracellular VEGFR signaling were used in the cell proliferation assay. HUVECs showed dose- and time-dependent proliferation decrease with all tested drugs over a 72 h incubation period. No- or hyper-VEGFR-methylation cancer cells showed no significant proliferation differences after treatment with VEGF-specific-antibody or VEGFR2-specific-antibody. After PTK/ZK or sunitinib treatment, no-VEGFR-methylation cancer cells showed dose- or time-dependent decreases in proliferation. Hyper-VEGFR-methylation cancer cells also showed proliferation inhibition by VEGF-specific-tyrosine kinase inhibitors after demethylation of Flt1 and KDR. Proliferation inhibition synergistically increased after combination of demethylation with PTK/ZK in hyper-VEGF-methylation cancer cells. We observed that intracellular targeting of VEGF-VEGFR signaling could be more effective than extracellular targeting of the pathway in the suppression of proliferation of some cancer cells. In particular, the efficacy of intracellular targeting of VEGF-specific-tyrosine kinase inhibitors might be influenced by the epigenetic alteration of VEGFRs.