Spectroscopic photoacoustic microscopic imaging during single spatial scan using broadband excitation light pulses with wavelength-dependent time delay.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.

An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation.

The centrosome lacks microtubule (MT)-nucleation activity in differentiated neurons. We have previously demonstrated that MTs were nucleated at the cytoplasm of mouse neurons. They are supposed to serve seeds for MTs required for dendrite growth. However, the factors that activate the cytoplasmic γ-tubulin ring complex (γTuRC) are unknown. Here we report an alternative splicing isoform of cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CKD5RAP2) as a candidate for the cytoplasmic γTuRC activator. This isoform lacked exon 17 and was expressed predominantly in the brain and testis. The expression was transient during the development of cortical neurons, which period coincided with the period we reported cytoplasmic MT nucleation. This isoform resulted in a frameshift and generated truncated protein without a centrosomal localization signal. When this isoform was expressed in cells, it localized diffusely in the cytoplasm. It was co-immunoprecipitated with γ-tubulin and MOZART2, suggesting that it can activate cytosolic γTuRCs. After cold-nocodazole depolymerization of MTs and subsequent washout, we observed numerous short MTs in the cytoplasm of cells transfected with the cDNA of this isoform. The isoform-overexpressing cells exhibited an increased amount of MTs and a decreased ratio of acetylated tubulin, suggesting that MT generation and turnover were enhanced by the isoform. Our data suggest the possibility that alternative splicing of CDK5RAP2 induces cytoplasmic nucleation of MTs in developing neurons.

A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy.

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues.

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes. Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated. Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs. Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through ß-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure.

Microencapsulated Sertoli cells sustain myoblast proliferation without affecting the myogenic potential. In vitro data.

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts.

The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties.

Background: Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for the development of novel therapeutic strategies. Here, we report in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (P2RX7B), in OS progression and metastasis. Methods: TE85 and MNNG-HOS OS cells were transfected with P2RX7B. These cell lines were then characterised and assessed for proliferation, cell adhesion, migration and invasion in vitro. We used these cells to perform both paratibial and tail vein injected mouse studies where the primary tumour, bone and lungs were analysed. We used RNA-seq to identify responsive pathways relating to P2RX7B. Results: Our data shows that P2RX7B expression confers a survival advantage in TE85 + P2RX7B and MNNG-HOS + P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, demonstrating a metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS + P2RX7B tumours exhibited cancer-associated ectopic bone formation that was abrogated with A740003 treatment. A pro-metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of tumour cells to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. Conclusion: Our data illustrates a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a future therapeutic target in human OS.

Gintonin influences the morphology and motility of adult brain neurons via LPA receptors.

BACKGROUND: Gintonin is an exogenous ginseng-derived G-protein-coupled lysophosphatidic acid (LPA) receptor ligand. LPA induces in vitro morphological changes and migration through neuronal LPA1 receptor. Recently, we reported that systemic administration of gintonin increases blood-brain barrier (BBB) permeability via the paracellular pathway and its binding to brain neurons. However, little is known about the influences of gintonin on in vivo neuron morphology and migration in the brain. MATERIALS AND METHODS: We examined the effects of gintonin on in vitro migration and morphology using primary hippocampal neural precursor cells (hNPC) and in vivo effects of gintonin on adult brain neurons using real time microscopic analysis and immunohistochemical analysis to observe the morphological and locational changes induced by gintonin treatment. RESULTS: We found that treating hNPCs with gintonin induced morphological changes with a cell rounding following cell aggregation and return to individual neurons with time relapses. However, the in vitro effects of gintonin on hNPCs were blocked by the LPA1/3 receptor antagonist, Ki16425, and Rho kinase inhibitor, Y27632. We also examined the in vivo effects of gintonin on the morphological changes and migration of neurons in adult mouse brains using anti-NeuN and -neurofilament H antibodies. We found that acute intravenous administration of gintonin induced morphological and migrational changes in brain neurons. Gintonin induced some migrations of neurons with shortened neurofilament H in the cortex. The in vivo effects of gintonin were also blocked by Ki16425. CONCLUSION: The present report raises the possibility that gintonin could enter the brain and exert its influences on the migration and morphology of adult mouse brain neurons and possibly explains the therapeutic effects of neurological diseases behind the gintonin administration.

Impact of molecular weight on the mechanism of cellular uptake of polyethylene glycols (PEGs) with particular reference to P-glycoprotein.

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g., PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (<2000 Da) occurs by passive diffusion whereas uptake of PEGs of high MW (>5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems.

Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8.

Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.

Consumption of Buglossoides arvensis seed oil is safe and increases tissue long-chain n-3 fatty acid content more than flax seed oil - results of a phase I randomised clinical trial.

Enrichment of tissues with ≥20-carbon n-3 PUFA like EPA is associated with positive cardiovascular outcomes. Stearidonic acid (SDA; 18 : 4n-3) and α-linolenic acid (ALA; 18 : 3n-3) are plant-derived dietary n-3 PUFA; however, direct comparisons of their impact on tissue n-3 PUFA content are lacking. Ahiflower(®) oil extracted from Buglossoides arvensis seeds is the richest known non-genetically modified source of dietary SDA. To investigate the safety and efficacy of dietary Ahiflower oil, a parallel-group, randomised, double-blind, comparator-controlled phase I clinical trial was performed. Diets of healthy subjects (n 40) were supplemented for 28 d with 9·1 g/d of Ahiflower (46 % ALA, 20 % SDA) or flax seed oil (59 % ALA). Blood and urine chemistries, blood lipid profiles, hepatic and renal function tests and haematology were measured as safety parameters. The fatty acid composition of fasting plasma, erythrocytes, polymorphonuclear cells and mononuclear cells were measured at baseline and after 14 and 28 d of supplementation. No clinically significant changes in safety parameters were measured in either group. Tissue ALA and EPA content increased in both groups compared with baseline, but EPA accrual in plasma and in all cell types was greater in the Ahiflower group (time × treatment interactions, P ≤ 0·01). Plasma and mononuclear cell eicosatetraenoic acid (20 : 4n-3) and docosapentaenoic acid (22 : 5n-3) content also increased significantly in the Ahiflower group compared with the flax group. In conclusion, the consumption of Ahiflower oil is safe and is more effective for the enrichment of tissues with 20- and 22-carbon n-3 PUFA than flax seed oil.

Glioblastoma cells induce differential glutamatergic gene expressions in human tumor-associated microglia/macrophages and monocyte-derived macrophages.

Glioblastoma cells produce and release high amounts of glutamate into the extracellular milieu and subsequently can trigger seizure in patients. Tumor-associated microglia/macrophages (TAMs), consisting of both parenchymal microglia and monocytes-derived macrophages (MDMs) recruited from the blood, are known to populate up to 1/3 of the glioblastoma tumor environment and exhibit an alternative, tumor-promoting and supporting phenotype. However, it is unknown how TAMs respond to the excess extracellular glutamate in the glioblastoma microenvironment. We investigated the expressions of genes related to glutamate transport and metabolism in human TAMs freshly isolated from glioblastoma resections. Quantitative real-time PCR analysis showed (i) significant increases in the expressions of GRIA2 (GluA2 or AMPA receptor 2), SLC1A2 (EAAT2), SLC1A3 (EAAT1), (ii) a near-significant decrease in the expression of SLC7A11 (cystine-glutamate antiporter xCT) and (iii) a remarkable increase in GLUL expression (glutamine synthetase) in these cells compared to adult primary human microglia. TAMs co-cultured with glioblastoma cells also exhibited a similar glutamatergic profile as freshly isolated TAMs except for a slight increase in SLC7A11 expression. We next analyzed these genes expressions in cultured human MDMs derived from peripheral blood monocytes for comparison. In contrast, MDMs co-cultured with glioblastoma cells compared to MDMs co-cultured with normal astrocytes exhibited decreased expressions in the tested genes except for GLUL. This is the first study to demonstrate transcriptional changes in glutamatergic signaling of TAMs in a glioblastoma microenvironment, and the findings here suggest that TAMs and MDMs might potentially elicit different cellular responses in the presence of excess extracellular glutamate.

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy.

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.

Involvement of angiotensin II type 2 receptor (AT2R) signaling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice.

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.

LIF-JAK1-STAT3 signaling delays contact inhibition of human corneal endothelial cells.

Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts.

Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.