Role of HIF1α Regulatory Factors in Stem Cells

Hypoxia-inducible factor 1 (HIF1) is a master transcription factor that induces the transcription of genes involved in the metabolism and behavior of stem cells. HIF1-mediated adaptation to hypoxia is required to maintain the pluripotency and survival of stem cells under hypoxic conditions. HIF1 activity is well known to be tightly controlled by the alpha subunit of HIF1 (HIF1α). Understanding the regulatory mechanisms that control HIF1 activity in stem cells will provide novel insights into stem cell biology under hypoxia. Recent research has unraveled the mechanistic details of HIF1α regulating processes, suggesting new strategies for regulating stem cells. This review summarizes recent experimental studies on the role of several regulatory factors (including calcium, 2-oxoglutarate-dependent dioxygenase, microtubule network, importin, and coactivators) in regulating HIF1α activity in stem cells.

Physiological understanding of host-microbial pathogen interactions in the gut

The gut epithelial barrier, which is composed of the mucosal layer and the intestinal epithelium, has multiple defense mechanisms and interconnected regulatory mechanisms against enteric microbial pathogens. However, many bacterial pathogens have highly evolved infectious stratagems that manipulate mucin production, epithelial cell-cell junctions, cell death, and cell turnover to promote their replication and pathogenicity in the gut epithelial barrier. In this review, we focus on current knowledge about how bacterial pathogens regulate mucin levels to circumvent the epithelial mucus barrier and target cell-cell junctions to invade deeper tissues and increase their colonization. We also describe how bacterial pathogens manipulate various modes of epithelial cell death to facilitate bacterial dissemination and virulence effects. Finally, we discuss recent investigating how bacterial pathogens regulate epithelial cell turnover and intestinal stem cell populations to modulate intestinal epithelium homeostasis.

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

Regulation of Stem Cell Fate by ROS-mediated Alteration of Metabolism

Stem cells have attracted much attention due to their distinct features that support infinite self-renewal and differentiation into the cellular derivatives of three lineages. Recent studies have suggested that many stem cells both embryonic and adult stem cells reside in a specialized niche defined by hypoxic condition. In this respect, distinguishing functional differences arising from the oxygen concentration is important in understanding the nature of stem cells and in controlling stem cell fate for therapeutic purposes. ROS act as cellular signaling molecules involved in the propagation of signaling and the translation of environmental cues into cellular responses to maintain cellular homeostasis, which is mediated by the coordination of various cellular processes, and to adapt cellular activity to available bioenergetic sources. Thus, in this review, we describe the physiological role of ROS in stem cell fate and its effect on the metabolic regulation of stem cells.

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit+ bone marrow cells (c-kit+ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit+ BM cells that give rise to CD2+CD8+ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit+ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2+CD8+ NK cells differentiated by cytokines from c-kit+ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2+CD8+ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit+ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit+ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

Multidetector computed tomographic angiography evaluation of micropig major systemic vessels for xenotransplantation

Due primarily to the increasing shortage of allogeneic donor organs, xenotransplantation has become the focus of a growing field of research. Currently, micropigs are the most suitable donor animal for humans. However, no standard method has been developed to evaluate the systemic vascular anatomy of micropigs and standard reference values to aid in the selection of normal healthy animals as potential organ donors are lacking. Using 64-channel multidetector row computed tomographic angiography (MDCTA), we evaluated morphological features of the major systemic vessels in micropigs and compared our results to published human data. The main vasculature of the animals was similar to that of humans, except for the iliac arterial system. However, diameters of the major systemic vessels were significantly different between micropigs and humans. Specifically, the diameter of the aortic arch, abdominal aorta, external iliac artery, and femoral artery, were measured as 1.50 +/- 0.07 cm, 0.85 +/- 0.06 cm, 0.52 +/- 0.05 cm, and 0.48 +/- 0.05 cm, respectively, in the micropigs. This MDCTA data for micropig major systemic vessels can be used as standard reference values for xenotransplantation studies. The use of 64-channel MDCTA enables accurate evaluation of the major systemic vasculature in micropigs.

The Role of Proximal Nephron in Cyclophosphamide-Induced Water Retention: Preliminary Data

Cyclophosphamide is clinically useful in treating malignancy and rheumatologic disease, but has limitations in that it induces hyponatremia. The mechanisms by which cyclophosphamide induces water retention in the kidney have yet to be identified. This study was undertaken to test the hypothesis that cyclophosphamide may produce water retention via the proximal nephron, where aquaporin-1 (AQP1) and aquaporin-7 (AQP7) water channels participate in water absorption. To test this hypothesis, we gave a single dose of intraperitoneal cyclophosphamide to male Sprague-Dawley rats and treated rabbit proximal tubule cells (PTCs) with 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide. In the short-term 3-day rat study, AQP1 protein expression was significantly increased in the whole kidney homogenates by cyclophosphamide administration at 48 (614 +/- 194%, P < 0.005), and 96 (460 +/- 46%, P < 0.05) mg/kg BW compared with vehicle-treated controls. Plasma sodium concentration was significantly decreased (143 +/- 1 vs. 146 +/- 1 mEq/L, P < 0.05) by cyclophosphamide 100 mg/kg BW in the long-term 6-day rat study. When primary cultured rabbit PTCs were treated with 4-HC for 24 hours, the protein expressions of AQP1 and AQP7 were increased in a dose-dependent manner. Quantitative polymerase chain reaction revealed no significant changes in the mRNA levels of AQP1 and AQP7 from cyclophosphamide-treated rat renal cortices. From these preliminary data, we conclude that the proximal nephron may be involved in cyclophosphamide-induced water retention via AQP1 and AQP7 water channels. Further studies are required to demonstrate intracellular mechanisms that affect the expression of AQP proteins.

High glucose stimulates glutamate uptakes in pancreatic beta-cells / 한국실험동물학회지

Pancreatic beta-cells are major cells responsible for glucose metabolism in the body. Hyperglycemia is known to be a primary factor in the induction of diabetes mellitus. Glutamate is also an excitatory neurotransmitter in diverse organs. Oxidative stress also plays a pivotal role in the development of diabetes mellitus. However, the effect of hyperglycemia in glutamate uptake in the pancreas is not clear. Furthermore, the relationship between high glucose-induced glutamate uptake and oxidative stress has not been investigated. Therefore, this study was conducted to investigate the effect of high glucose on glutamate uptake in pancreatic beta-cells. In the present study, 25 mM glucose stimulated the glutamate uptake in HIT-15 cells of hamster pancreatic beta-cells. The treatment of 25 mM glucose and 1 mM glutamate also decreased the cell viability in HIT-15 cells. In addition, the treatment of 25 mM glucose induced an increase of lipid peroxide formation. High glucose-induced increase of LPO formation was prevented by the treatment of antioxidants such as N-acetyl-L-cysteine and quercetin. Furthermore, high glucose-induced stimulation of glutamate uptake and decrease of cell viability were also blocked by the treatment of N-acetyl-L-cysteine and quercetin. In conclusion, high glucose stimulated glutamate uptake via oxidative stress in pancreatic beta-cells.

Magnetic resonance imaging evaluation of Yukatan minipig brains for neurotherapy applications / 한국실험동물학회지

Magnetic resonance imaging (MRI) of six Yukatan minipig brains was performed. The animals were placed in stereotaxic conditions currently used in experiments. To allow for correctpositioning of the animal in the MRI instrument, landmarks were previously traced on the snout of the pig. To avoid movements, animal were anesthetized. The animals were placed in a prone position in a Siemens Magnetom Avanto 1.5 System with a head coil. Axial T2-weighted and sagittal T1-weighted MRI images were obtained from each pig. Afterwards, the brains of the pigs were fixed and cut into axial sections. Histologic and MR images were compared. The usefulness of this technique is discussed.

Tissue Specific Expression of Lipid Metabolism Related Molecules in Digestive Organs of Miniature Pigs / 한국실험동물학회지

The miniature pig is a very suitable donor species in xenotransplantation of human organs. Lipid metabolism is an important process that involves the creation and degradation of lipids, which is associated with the function of the gastro-intestinal tract. However, the distribution of lipid metabolism related molecules in the gastro-intestinal tract in the miniature pig is unclear. The present study examined the expression of farnesoid X-receptor (FXR), liver X- receptor (LXR), retinoid X-receptor (RXR), liver fatty acid binding protein (L-FABP), fatty acid synthase (FAS) mRNA in the digestive organs of miniature pigs. FXR and LXR mRNA were not expressed in the stomach but were expressed at high and low density in the small and large intestines, respectively. RXR mRNA was expressed in stomach with moderate density, small intestine with high density and in the large intestine with low density. L-FABP and FAS mRNA were expressed in the stomach and large intestine with low density and in the small intestine with high density. L-FABP mRNA was expressed in the liver and kidney with high density, and in pancreas with low density. FAS mRNA was expressed in the liver with high density, and in pancreas and kidney with low density.

Tissue Specific Expression of Lipid Metabolism Related Molecules in Digestive Organs of Miniature Pigs / 한국실험동물학회지

The miniature pig is a very suitable donor species in xenotransplantation of human organs. Lipid metabolism is an important process that involves the creation and degradation of lipids, which is associated with the function of the gastro-intestinal tract. However, the distribution of lipid metabolism related molecules in the gastro-intestinal tract in the miniature pig is unclear. The present study examined the expression of farnesoid X-receptor (FXR), liver X- receptor (LXR), retinoid X-receptor (RXR), liver fatty acid binding protein (L-FABP), fatty acid synthase (FAS) mRNA in the digestive organs of miniature pigs. FXR and LXR mRNA were not expressed in the stomach but were expressed at high and low density in the small and large intestines, respectively. RXR mRNA was expressed in stomach with moderate density, small intestine with high density and in the large intestine with low density. L-FABP and FAS mRNA were expressed in the stomach and large intestine with low density and in the small intestine with high density. L-FABP mRNA was expressed in the liver and kidney with high density, and in pancreas with low density. FAS mRNA was expressed in the liver with high density, and in pancreas and kidney with low density.

Multidetector row computed tomography evaluation of the micropig kidney as a potential renal donor

Multidetector row computed tomography (MDCT) provides anatomical information about the kidney and other internal organs. Presently, the suitability of 64-channel MDCT to assess the kidney of healthy micropigs was evaluated. Morphological evaluations of the kidney and the major renal vessels of six healthy micropigs were carried out using MDCT, recording kidney volume and the diameter and length of renal arteries and veins. The mean diameters and lengths of the renal artery were 0.44 +/- 0.05 and 4.51 +/- 0.55 cm on the right side and 0.46 +/- 0.06 and 3.36 +/- 0.27 cm on the left side, respectively. The mean diameters and lengths of the renal vein were 1.44 +/- 0.52 and 4.22 +/- 1.29 cm on the right side and 1.38 +/- 0.17 and 5.15 +/- 0.87 cm on the left side, respectively. The mean volume of the right kidney was 79.3 +/- 14.5 mL and of the left kidney was 78.0 +/- 13.9 mL. The data presented in this study suggest that the MDCT offers a noninvasive, rapid, and accurate method for the evaluation of the renal anatomy in living kidney donors. It also provides sufficient information about extra-renal anatomy important for donor surgery and determination of organ suitability.

64-Channel multi-detector row CT angiographic evaluation of the micropigs for potential living donor lung transplantation

Micropigs are the most likely source animals for xenotransplantation. However, an appropriate method for evaluating the lung of micropigs had not been established. Therefore, this study was performed to evaluate the feasibility of 64-channel multi-detector row computed tomography (MDCT) to measure the diameter of the pulmonary arteries and the lung volume in micropigs. The mean diameters of the trachea, and left and right bronchi were 1.6 +/- 0.17, 1.18 +/- 0.14, and 1.1 +/- 0.11 cm, respectively. The mean diameters of the main, right, and left pulmonary arteries were 1.38 +/- 0.09, 1.07 +/- 0.26, and 0.98 +/- 0.13 cm and the diameters of right, left, and common inferior pulmonary veins were 0.97 +/- 0.20, 0.76 +/- 0.20, and 1.99 +/- 0.26 cm, respectively. The mean lung volume was 820.3 +/- 77.11 mL. The data presented in this study suggest that the MDCT may be a noninvasive, rapid, and accurate investigational method for pulmonary anatomy in living lung donors.

Scintigraphic Assessment of Renal Function Using 99mTc-DTPA in Miniature Pigs with Unilateral Ureteral Obstruction / 한국실험동물학회지

This study was performed to investigate the proper method for evaluating renal function in miniature pigs with unilateral ureteral obstruction. Experimental unilateral renal damage was induced after ligation of unilateral right ureter in 3 miniature pigs. On the 3rd post-operative day, scintigraphic images were obtained after 12 mCi of 99mTc-diethylentriamene pertaacetate (DTPA) intravenous injection. Renography showed that radiopharmaceutical uptakes in the right kidney were lower than those of left kidney uptakes as early as at 3 days after surgical operation. The static images of 99mTc-DTPA enabled us to measure the relative renal function in miniature pigs with unilateral ureteral obstruction. In conclusion, renography using 99mTc-DTPA was the useful diagnostic method to evaluate the renal function in miniature pigs.

Imaging evaluation of the liver using multi-detector row computed tomography in micropigs as potential living liver donors

The shortage of organ donors has stimulated interest in the possibility of using animal organs for transplantation into humans. In addition, pigs are now considered to be the most likely source animals for human xenotransplantation because of their advantages over non-human primates. However, the appropriate standard values for estimations of the liver of micropigs have not been established. The determination of standard values for the micropig liver using multi-detector row computed tomography (MDCT) would help to select a suitable donor for an individual patient, determine the condition of the liver of the micropigs and help predict patient prognosis. Therefore, we determined the standard values for the livers of micropigs using MDCT. The liver parenchyma showed homogenous enhancement and had no space-occupying lesions. The total and right lobe volumes of the liver were 698.57 +/- 47.81 ml and 420.14 +/- 26.70 ml, which are 51.74% and 49.35% of the human liver volume, respectively. In micropigs, the percentage of liver volume to body weight was approximately 2.05%. The diameters of the common hepatic artery and proper hepatic artery were 6.24 +/- 0.20 mm and 4.68 +/- 0.13 mm, respectively. The hepatic vascular system of the micropigs was similar to that of humans, except for the variation in the length of the proper hepatic artery. In addition, the diameter of the portal vein was 11.27 +/- 0.38 mm. In conclusion, imaging evaluation using the MDCT was a reliable method for liver evaluation and its vascular anatomy for xenotransplantation using micropigs.

Comparison of cardiac function and coronary angiography between conventional pigs and micropigs as measured by multidetector row computed tomography

Pigs are the most likely source animals for cardiac xenotransplantation. However, an appropriate method for estimating the cardiac function of micropigs had not been established. Computed tomography (CT) analysis aimed at estimating cardiac function and assessing the coronary arteries has not been carried out in micropigs. This study determined the feasibility of evaluating cardiac function in a micropig model using multidetector row computed tomography (MDCT) and compared the cardiac function values with those of conventional pigs. The mean age of the conventional pigs and micropigs was approximately 80 days and approximately 360 days, respectively. The mean body weight in the conventional pigs and micropigs was 29.70 +/- 0.73 and 34.10 +/- 0.98 kg, respectively. Cardiac MDCT detected ejection fractions of 52.93 +/- 3.10% and 59.00 +/- 5.56% and cardiac outputs of 1.46 +/- 0.64 l/min and 1.21 +/- 0.24 l/min in conventional pigs and micropigs, respectively. There were no significant differences in cardiac function between conventional pigs and micropigs in the reconstructed CT images. There were also no differences in the coronary angiographic images obtained by MDCT. It is expected that the results of this study will help improve understanding of cardiac function in micropigs. The data presented in this study suggest that MDCT is a feasible method for evaluating cardiac function in micropigs.

Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H(2)O(2)-induced oxidative stress

Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5alpha- reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10(-7) M) on H(2)O(2) (10(-3) M) -induced injuries in mouse embryonic stem (ES) cells. H(2)O(2) induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H(2)O(2) were inhibited by pretreatment with DHT. H(2)O(2) also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-kappaB), but DHT blocked these effects. Moreover, H(2)O(2) decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H(2)O(2) were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H(2)O(2)-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-kappaB in mouse ES cells.

Effects of Intracavernosally Injected Embryonic Stem Cells in Aged Rat / 대한남성과학회지

PURPOSE: Stem cell-based cell therapy has recently been tried as a way to restore cavernosal function in an animal model. The aims of this study were to elucidate the effect of intracavernosally injected embryonic stem cells(ESCs) in aged rat. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into 3 groups: young control(12 weeks old; n=10), old with vehicle injection(24 months old; n=7), and old with ESC injection(24 months old; n=8). ESCs were transfected with firefly luciferase attached to adenovirus and then injected intracavernously 2 times with a 1-week interval. Cell survival was assessed by optical molecular imaging 2 days after the last ESC injection. At 4 weeks after the last injection, intracavernosal pressure and systemic arterial pressure were recorded after pelvic nerve stimulation. Serum testosterone levels were measured by radioimmunoassay. RESULTS: We observed fluorescent signals around the external genitalia of animals injected with ESCs. The serum testosterone level of the old group(1.39+/-0.07 ng/ml) was significantly lower compared to the young control group(2.98+/-0.31 ng/ml)(p=0.03). The percentage of intracavernosal pressure/systolic blood pressure was significantly lower in the old group(58.5+/-8.6%) compared to the young control group(69.5+/-6.6%)(p=0.034). However, the old group with ESC injection(61.6+/-9.9%) did not show any significant differences from the old group(p>0.05). The old group with ESC injection showed histomorphometry similar to the old group. CONCLUSIONS: The presence of intracavernosal ESCs can be noninvasively monitored with optical molecular imaging. However, the intracavernosal injection of ESCs did not improve erectile function in the aging rat. Further studies are needed to elucidate the regulatory factors of stem cell differentiation in the corpus cavernosum.

Expression and Translocation of Aquaporin (AQP)-1 after Pelvic Nerve Stimulation in Rat Vagina / 대한남성과학회지

PURPOSE: Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. The purposes of this study were to investigate the localization and functional roles of AQP-1 water channels in rat vagina. MATERIALS AND METHODS: Female Sprague-Dawley rats (230~240 g, n=40) were anesthetized. To investigate the expression and localization of AQPs in the vagina, the vaginal branch of the pelvic nerve was stimulated for 60 seconds (10 V, 16 Hz, 0.8 msec), and then the animals were sacrificed immediately or 5 minutes later. The expression and cellular localization of AQP-1 in the vaginal tissue was measured by Western blot and immunohistochemistry. The cytosolic (or intracellular membrane) and plasma membrane fractions of AQP-1 in vaginal tissue were studied by immunoblot analysis. RESULTS: Immunolabeling showed that AQP-1 was mainly expressed in the capillaries and venules of the vagina. The translocation of AQP-1 isoforms from the cytosolic compartment to the plasma membrane compartment was observed right after nerve stimulation and had declined at 5 minutes after nerve stimulation. However, when the nerve stimulation was repeated 3 times, the translocation of AQP-1 from the intracellular membrane compartment to the plasma membrane compartment was still observed at 5 minutes. CONCLUSIONS: This study suggests that sexual arousal induced by pelvic nerve stimulation modulates AQP-1 activity in the rat vagina. This result implies that AQP-1 may play an important role in vaginal lubrication.

Effectiveness of 99mTc-tetrofosmin for assessment of heart functions in micropigs

This study examined the suitability of a nuclear imagingtechnique using 99mTc-tetrofosmin as an agent to assess theheart functions of healthy micropigs. The mean age of thepigs was 360 days (male), and the mean body weight was35.3kg ranging from 34.5-36kg. There were no significantperfusion defects in any of the reconstructed images.Gated single-photon emission computed tomographyimaging can be used to calculate the ventricular volumeand ejection fraction (EF). In this case, an EF of 79% wascalculated from the ventricular volume of the end-systolicimage (10 ml) subtracted from that of the end-diastolicvolume (49 ml). A perfusion defect (particularly the apex,lateral wall) is unlikely because of the presence of apreserved wall motion in a segment with a defect. It isconcluded that quantitative cardiac scintigraphy, using99mTc-tetrofosmin is an adequate technique for estimatingthe heart functions of healthy micropigs.