DNA-PKcs and ATM modulate mitochondrial ADP-ATP exchange as an oxidative stress checkpoint mechanism.

DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis.

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Transcriptional regulation of lysophosphatidic acid receptors 2 and 3 regulates myeloid commitment of hematopoietic stem cells.

Lysophosphatidic acid (LPA) is one of the lipids identified to be involved in stem cell differentiation. It exerts various functions through activation of G protein-coupled lysophosphatidic acid receptors (LPARs). In previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis of human hematopoietic stem cells (HSCs) and zebrafish using molecular and pharmacological approaches. Our results show that treatment with lysophosphatidic acid receptor 2 (LPA2) agonist suppressed erythropoiesis, whereas activation of LPA3 by 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted it, both in vitro and in vivo. Furthermore, we have demonstrated the inhibitory role of LPA3 during megakaryopoiesis. However, the mechanism underlying these observations remains elusive. In the present study, we suggest that the expression pattern of LPARs may be correlated with the transcriptional factors GATA-1 and GATA-2 at different stages of myeloid progenitors. We determined that manipulation of GATA factors affected the expression levels of LPA2 and LPA3 in K562 leukemia cells. Using luciferase assays, we demonstrate that the promoter regions of LPAR2 and LPAR3 genes were regulated by these GATA factors in HEK293T cells. Mutation of GATA-binding sites in these regions abrogated luciferase activity, suggesting that LPA2 and LPA3 are regulated by GATA factors. Moreover, physical interaction between GATA factors and the promoter region of LPAR genes was verified in K562 cells using chromatin immunoprecipitation (ChIP) studies. Taken together, our results suggest that balance between LPA2 and LPA3 expression, which may be determined by GATA factors, is a regulatory switch for lineage commitment in myeloid progenitors. The expression-level balance of LPA receptor subtypes represents a novel mechanism regulating erythropoiesis and megakaryopoiesis.

Phosphatidylinositol-4-phosphate 5-kinase type 1α attenuates Aß production by promoting non-amyloidogenic processing of amyloid precursor protein.

Alzheimer's disease (AD) is characterized by a chronic decline in cognitive function and is pathologically typified by cerebral deposition of amyloid-ß peptide (Aß). The production of Aß is mediated by sequential proteolysis of amyloid precursor protein (APP) by ß- and γ-secretases, and has been implicated as the essential determinant of AD pathology. Previous studies have demonstrated that the level of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in the membrane may potentially modulate Aß production. Given that PI(4,5)P2 is produced by type 1 phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), we sought to determine whether the level of PIP5K type Iα (PIP5K1A) can affect production of Aß by modulating the lipid composition of the membrane. Using a HEK-derived cell line that constitutively expresses yellow fluorescent protein-tagged APP (APP-YFP), we demonstrated that overexpression of PIP5K1A results in significant enhancement of non-amyloidogenic APP processing and a concomitant suppression of the amyloidogenic pathway, leading to a marked decrease in secreted Aß. Consistently, cells overexpressing PIP5K1A exhibited a significant redistribution of APP-YFP from endosomal compartments to the cell surface. Our findings suggest that PIP5K1A may play a critical role in governing Aß production by modulating membrane distribution of APP, and as such, the pathway may be a valuable therapeutic target for AD.

PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway.

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.

Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]- and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-ß in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.

Lysophosphatidic Acid and Hematopoiesis: From Microenvironmental Effects to Intracellular Signaling.

Vertebrate hematopoiesis is a complex physiological process that is tightly regulated by intracellular signaling and extracellular microenvironment. In recent decades, breakthroughs in lineage-tracing technologies and lipidomics have revealed the existence of numerous lipid molecules in hematopoietic microenvironment. Lysophosphatidic acid (LPA), a bioactive phospholipid molecule, is one of the identified lipids that participates in hematopoiesis. LPA exhibits various physiological functions through activation of G-protein-coupled receptors. The functions of these LPARs have been widely studied in stem cells, while the roles of LPARs in hematopoietic stem cells have rarely been examined. Nonetheless, mounting evidence supports the importance of the LPA-LPAR axis in hematopoiesis. In this article, we have reviewed regulation of hematopoiesis in general and focused on the microenvironmental and intracellular effects of the LPA in hematopoiesis. Discoveries in these areas may be beneficial to our understanding of blood-related disorders, especially in the context of prevention and therapy for anemia.

A Novel Function of the Lysophosphatidic Acid Receptor 3 (LPAR3) Gene in Zebrafish on Modulating Anxiety, Circadian Rhythm Locomotor Activity, and Short-Term Memory.

Lysophosphatidic acid (LPA) is a small lysophospholipid molecule that activates multiple cellular functions through pathways with G-protein-coupled receptors. So far, six LPA receptors (LPAR1 to LPAR6) have been discovered and each one of them can connect to the downstream cell message-transmitting network. A previous study demonstrated that LPA receptors found in blood-producing stem cells can enhance erythropoietic processes through the activation of LPAR3. In the current study, newly discovered functions of LPAR3 were identified through extensive behavioral tests in lpar3 knockout (KO) zebrafish. It was found that the adult lpar3 KO zebrafish display an abnormal movement orientation and altered exploratory behavior compared to that of the control group in the three-dimensional locomotor and novel tank tests, respectively. Furthermore, consistent with those results, in the circadian rhythm locomotor activity test, the lpar3 KO zebrafish showed a lower level of angular velocity and average speed during the light cycles, indicating an hyperactivity-like behavior. In addition, the mutant fish also exhibited considerably higher locomotor activity during the dark cycle. Supporting those findings, this phenomenon was also displayed in the lpar3 KO zebrafish larvae. Furthermore, several important behavior alterations were also observed in the adult lpar3 KO fish, including a lower degree of aggression, less interest in conspecific social interaction, and looser shoal formation. However, there was no significant difference regarding the predator avoidance behavior between the mutant and the control fish. In addition, lpar3 KO zebrafish displayed memory deficiency in the passive avoidance test. These in vivo results support for the first time that the lpar3 gene plays a novel role in modulating behaviors of anxiety, aggression, social interaction, circadian rhythm locomotor activity, and memory retention in zebrafish.

Intentional endometrial injury increases embryo implantation potentials through enhanced endometrial angiogenesis†.

Embryo implantation rates have been found to be enhanced by precedent endometrial injuries, but the underlying mechanism is not fully investigated. Endometrial inflammation occurs both at peri-implantation period and after endometrial injury, in which vascular reaction is a distinctive feature of inflammation. In this study, intentional endometrial injury was done with a 0.7-mm-diameter brush inserted into the left uterine horn of female ICR mice, then turned around 720° (group 2), and the right uterine horn served as the controls without endometrial injuries (group 1). Intraperitoneal equine chorionic gonadotropin 2.5 IU was injected, followed by human chorionic gonadotropin 10 IU injection, and the uterus was dissected 5 days later, roughly at the peri-implantation period. The peri-implantation endometrium was obtained, and angiogenesis protein array revealed that matrix metalloproteinase-3 (MMP-3), plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor binding protein 1 (IGFBP-1), and IL-1α were more strongly expressed in injured endometrium (group 2) than in the controls (group 1). Immunohistochemical CD34 staining was more prominently expressed in group 2 uterus, and the treatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly decreased CD34 immunopositive cells. The capabilities of permeability, proliferation, tube formation, and migration of mouse endometrial endothelial cells were significantly enhanced in group 2 than in group 1. Our results demonstrate that enhanced endometrial angiogenesis is a possible mechanism accounting for the increased endometrial receptivity after endometrial injury.

Growth of heterojunctions in Si-Ge alloy nanowires by altering AuGeSi eutectic composition using an approach based on thermal oxidation.

Understanding the growth mechanism of heterojunctions in silicon-germanium alloy (Si-Ge) nanowires is helpful for designing adequate physical properties in the material for device applications. We examine the formation of the heterojunction in low Ge-content Si-Ge nanowires by an approach of thermal oxidation, which produces an atomically abrupt interface with an obvious concentration change. Forming heterojunctions in Si-Ge nanowires by this approach involves more complicated reaction routes than direct growth of heterojunction nanowires using the vapor-liquid-solid method. At the beginning of the oxidation process, the AuGeSi eutectic liquid at the nanowire tip significantly etches the Si-Ge alloy nanowires. Selective oxidation of Si results in a change of the relative amount of Ge to Si in the eutectic liquid, which further modulates the solubility of Ge and Si atoms. The compositional variation in the Au-Ge-Si ternary alloy system during the oxidation process accounts for the observed concentration profile in the heterojunction nanowire. The thermal oxidation approach is applied on a low Ge-content Si-Ge thin film that is coated with Au nanoparticles. Si-Ge nanodots, which exhibit a higher Ge concentration, are precipitated epitaxially in the film, as a result of compositional modulation in the AuGeSi eutectic liquid.

The Antithrombotic Function of Sphingosine-1-Phosphate on Human Adipose-Stem-Cell-Recellularized Tissue Engineered Vascular Graft In Vitro.

Adipose stem cells (ASCs) show potential in the recellularization of tissue engineerined vascular grafts (TEVGs). However, whether sphingosine-1-phosphate (S1P) could further enhance the adhesion, proliferation, and antithrombosis of ASCs on decellularized vascular scaffolds is unknown. This study investigated the effect of S1P on the recellularization of TEVGs with ASCs. Human ASCs were derived from lipoaspirate. Scaffolds were derived from human umbilical arteries (HUAs) with treatment of 0.1% sodium dodecyl sulfate (SDS) for 48 h (decellularized HUAs; DHUAs). The adhesion, proliferation, and antithrombotic functions (kinetic clotting time and platelet adhesion) of ASCs on DHUAs with S1P or without S1P were evaluated. The histology and DNA examination revealed a preserved structure and the elimination of the nuclear component more than 95% in HUAs after decellularizaiton. Human ASCs (hASCs) showed CD29(+), CD73(+), CD90(+), CD105(+), CD31(-), CD34(-), CD44(-), HLA-DR(-), and CD146(-) while S1P-treated ASCs showed marker shifting to CD31(+). In contrast to human umbilical vein endothelial cells (HUVECs), S1P didn't significantly increase proliferation of ASCs on DHUAs. However, the kinetic clotting test revealed prolonged blood clotting in S1P-treated ASC-recellularized DHUAs. S1P also decreased platelet adhesion on ASC-recellularized DHUAs. In addition, S1P treatment increased the syndecan-1 expression of ASCs. TEVG reconstituted with S1P and ASC-recellularized DHUAs showed an antithrombotic effect in vitro. The preliminary results showed that ASCs could adhere to DHUAs and S1P could increase the antithrombotic effect on ASC-recellularized DHUAs. The antithrombotic effect is related to ASCs exhibiting an endothelial-cell-like function and preventing of syndecan-1 shedding. A future animal study is warranted to prove this novel method.

Sphingosine-1-phosphate in Endothelial Cell Recellularization Improves Patency and Endothelialization of Decellularized Vascular Grafts In Vivo.

BACKGROUND: S1P has been shown to improve the endothelialization of decellularized vascular grafts in vitro. Here, we evaluated the potential of tissue-engineered vascular grafts (TEVGs) constructed by ECs and S1P on decellularized vascular scaffolds in a rat model. METHODS: Rat aorta was decellularized mainly by 0.1% SDS and characterized by histology. Rat ECs, were seeded onto decellularized scaffolds, and the viability of the ECs was evaluated by biochemical assays. Then, we investigated the in vivo patency rate and endothelialization for five groups of decellularized vascular grafts (each n = 6) in a rat abdominal aorta model for 14 days. The five groups included (1) rat allogenic aorta (RAA); (2) decellularized RAA (DRAA); (3) DRAA with S1P (DRAA/S1P); (4) DRAA with EC recellularization (DRAA/EC); and (5) DRAA with S1P and EC recellularization (DRAA/EC/S1P). RESULTS: In vitro, ECs were identified by the uptake of Dil-Ac-LDL. S1P enhanced the expression of syndecan-1 on ECs and supported the proliferation of ECs on decellularized vascular grafts. In vivo, RAA and DRAA/EC/S1P both had 100% patency without thrombus formation within 14 days. Better endothelialization, more wall structure maintenance and less inflammation were noted in the DRAA/EC/S1P group. In contrast, there was thrombus formation in the DRAA, DRAA/S1P and DRAA/EC groups. CONCLUSION: S1P could inhibit thrombus formation to improve the patency rate of EC-covered decellularized vascular grafts in vivo and may play an important role in the construction of TEVGs.

High Glucose Induces VEGF-C Expression via the LPA1/3-Akt-ROS-LEDGF Signaling Axis in Human Prostate Cancer PC-3 Cells.

BACKGROUND/AIMS: Hyperglycemia has been shown to increase the incidence and metastasis in various types of cancers. However, the correlation between hyperglycemia and lymphatic metastasis in prostate cancer (PCa) remains unclear. Our previous study demonstrated that lysophosphatidic acid (LPA) enhances vascular endothelial growth factor-C (VEGF-C) expression, a lymphangiogenic factor, through activating it receptors LPA1/3 in prostate cancer (PCa) cells. Moreover, hyperglycemia up-regulates autotaxin (ATX) expression, a LPA-generating enzyme. Therefore, we propose that high glucose promotes VEGF-C expression through LPA signaling in PCa cells. METHODS: Pharmacological inhibitors and siRNAs were utilized to investigate the molecular mechanism of high glucose-induced VEGF-C expression. Real-time PCR and Western blot were used to determine the mRNA and protein expressions, respectively. Cellular bioenergetics analysis was performed to determine the glycolysis levels. RESULTS: We demonstrated that the expressions of VEGF-C, ATX, and calreticulin were increased upon high glucose treatments in PC-3 cells. Moreover, high glucose-induced VEGF-C expression was mediated through the LPA1/3, PLC, Akt, ROS and LEDGF-dependent pathways. Additionally, high glucose enhanced the aerobic glycolysis via LPA1/3. CONCLUSION: These results indicated that hyperglycemia leads to LPA synthesis, and subsequent promoting pathological consequence of PCa. These novel findings could potentially provide new strategies for PCa treatments.

Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for target engagement challenges in situ.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide using S-adenosyl-L-methionine (SAM) as a methyl donor and, through doing so, can modulate cellular methylation potential to impact diverse epigenetic processes. NNMT has been implicated in a range of diseases, including cancer and metabolic disorders. Potent, selective, and cell-active inhibitors would constitute valuable probes to study the biological functions and therapeutic potential of NNMT. We previously reported the discovery of electrophilic small molecules that inhibit NNMT by reacting with an active-site cysteine residue in the SAM-binding pocket. Here, we have used activity-based protein profiling (ABPP)-guided medicinal chemistry to optimize the potency and selectivity of NNMT inhibitors, culminating in the discovery of multiple alpha-chloroacetamide (αCA) compounds with sub-µM IC50 values in vitro and excellent proteomic selectivity in cell lysates. However, these compounds showed much weaker inhibition of NNMT in cells, a feature that was not shared by off-targets of the αCAs. Our results show the potential for developing potent and selective covalent inhibitors of NNMT, but also highlight challenges that may be faced in targeting this enzyme in cellular systems.

Producing Atomically Abrupt Axial Heterojunctions in Silicon-Germanium Nanowires by Thermal Oxidation.

Compositional abruptness of the interfaces is one of the important factors to determine the performance of Group IV semiconductor heterojunction (Si/Ge or Si/SiGe) nanowire devices. However, forming abrupt interfaces in the nanowires using the common vapor-liquid-solid (VLS) method is restricted because large solubility of Si and Ge in the Au eutectic liquid catalyst makes gradual composition change at the heterojunction after switching the gas phase components. According to the VLS growth mechanism, another possible approach to form an abrupt interface is making a change of the semiconductor concentration in the eutectic liquid before precipitation of the second phase. Here we show that the composition in AuSiGe eutectic liquid on SiGe nanowires of low Ge concentration (≤6%) can be altered by thermal oxidation at 700 °C. During the oxidation process, only Si is oxidized on the surface of the eutectic liquid, and the Ge/Si ratio in the eutectic liquid is increased. The subsequently precipitated SiGe step at the liquid/solid interface has a higher Ge concentration (∼20%), and a compositionally abrupt interface is produced in the nanowires. The growth mechanism of the heterojunction includes diffusion of Si and Ge atoms on nanowire surface into the AuSiGe eutectic liquid and step nucleation at the liquid/nanowire interface.

In Vivo Performance of Decellularized Vascular Grafts: A Review Article.

Due to poor vessel quality in patients with cardiovascular diseases, there has been an increased demand for small-diameter tissue-engineered blood vessels that can be used as replacement grafts in bypass surgery. Decellularization techniques to minimize cellular inflammation have been applied in tissue engineering research for the development of small-diameter vascular grafts. The biocompatibility of allogenic or xenogenic decellularized matrices has been evaluated in vitro and in vivo. Both short-term and long-term preclinical studies are crucial for evaluation of the in vivo performance of decellularized vascular grafts. This review offers insight into the various preclinical studies that have been performed using decellularized vascular grafts. Different strategies, such as surface-modified, recellularized, or hybrid vascular grafts, used to improve neoendothelialization and vascular wall remodeling, are also highlighted. This review provides information on the current status and the future development of decellularized vascular grafts.

Opposing regulation of megakaryopoiesis by LPA receptors 2 and 3 in K562 human erythroleukemia cells.

Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Lysophosphatidic acid (LPA), a lipid growth factor was previously shown to be a regulator for erythropoietic process through activating LPA receptor 3 (LPA3). However, whether LPA affects megakaryopoiesis remains unclear. In this study, we used K562 leukemia cell line as a model to investigate the roles of LPA in MK differentiation. We demonstrated that K562 cells express both LPA2 and LPA3, and the expression levels of LPA2 are higher than LPA3. Treatment with phorbol 12-myristate 13-acetate, a commonly used inducer of megakaryopoiesis, reciprocally regulates the expressions of LPA2 and LPA3. By pharmacological blockers and knockdown experiments, we showed that activation of LPA2 suppresses whereas, LPA3 promotes megakaryocytic differentiation in K562. The LPA2-mediated inhibition is dependent on ß-catenin translocation, whereas reactive oxygen species (ROS) generation is a downstream signal for activation of LPA3. Furthermore, the hematopoietic transcriptional factors GATA-1 and FLI-1, appear to be involved in these regulatory mechanisms. Taken together, our results suggested that LPA2 and LPA3 may function as a molecular switch and play opposing roles during megakaryopoiesis of K562 cells.