BrdU Uptake Research Articles

The increase in thymopoiesis following T cell progenitor therapy is dependent upon the input population and continued interaction between developing T cells and the thymic microenvironment.

Abstract The inclusion of in vitro derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant (HSCT) aids in the recovery of the thymus damaged by total body irradiation and improves de novo thymopoiesis. To understand the interaction between proTs and the thymic microenvironment, wildtype (WT) mice were lethally irradiated and given T cell deficient donor (Rag1−/−) marrow along with in vitro generated proT from WT donors, limiting mature T cell development to infused proT. Donor proTs within the host thymus led to a significant increase in thymic epithelial cell (TEC) numbers by day 21 post-transplant, and increased actively cycling TECs as measured by Ki67 expression and BrdU uptake. However, that gain was temporary and lost by day 28, suggesting that continued signaling from proT cells is required to sustain TEC cycling and mass. We also find a significant improvement in total thymocyte number by day 21 followed by a significant increase in the total mature T cell number in the secondary lymphoid organs by day 28. This protective surge is also temporary, receding by day 60. In this time period, infused DN2 proTs selectively increased thymocyte number while DN3 proTs preferentially led to a greater TEC numbers. Interestingly, an exception in persistence occurs when DN3 proTs are used and the increase in mature T cells in the spleen persists at day 60. As a result of the lack of competition for thymic niches by cells from the Rag1−/− graft, a subpopulation of the infused proT persisted in the thymus in an immature state at day 60. These findings highlight the importance of the interaction between developing T cells and TECs in the proliferation and survival of these critical components of the thymic microenvironment.

High Monocyte Turnover Predicts Progression to AIDS in SIV Infected Aging Rhesus Macaques

Abstract Incidence of newly-acquired HIV infections in persons over age 50 is increasing, from 13% in 2006 to 21% in 2013. Advanced age corresponds with lower 12-month survival and ART is contraindicated with the simultaneous use of many chronic disease medications. To address the need for age-specific interventions, we are studying aged rhesus macaques (RM) infected with SIV. In a pilot study, four young (6.27–7.37 years) and six older (17.71–23.82 years) RM were injected i.v. with SIVmac239. The older RM exhibited lower survival times compared to younger macaques (112.5 ± 76.8 days v. 353 ± 74.9 days, respectively). In young adult RM with SIV, we reported that increasing monocyte turnover (via BrdU uptake) as well as declining CD4+ cell counts, predicted disease progression. Among the aged RM, we observed that 3 animals developed increased or high monocyte turnover (HMT) after the first month of infection (35.2% ± 2.36) while the other 3 maintained steady state or low monocyte turnover (LMT) (5.74% ± 4.92) similar to that in the young animals (9.97% ± 2.34). Older RM with LMT survived longer (151 ± 85.6 days) than those with HMT (74 ± 54.5 days). The young adult and aged RM with LMT exhibited a dramatic decline of CD4+ T cells during peak viral load (−46% and −41%) 2 wks post infection compared to only a 22% decrease in aged RM with HMT. Also, the numbers of naïve T cells (CD4 and CD8) were lower in the HMT aged animals than in the LMT and young adults, at baseline and 2 wks post infection. These results suggest that there may be an additional or alternative preferential host cell target of SIV early in infection of older susceptible RM that we speculate to be macrophages. Work is continuing to examine the role of macrophages in pathogenesis of HIV infection in the elderly.

Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist

Abstract Signaling lymphocyte activation marker family member 6 (Slamf6) is a cell surface signaling receptor that plays an important role in NKT cell development. Upon activation with the CD1d ligand α-galactosylceramide (αGalCer), NKT cell Slamf6 expression increases dramatically (~10-fold) and it maintains this high level of expression for at least 5 days after activation. The mechanisms through which Slamf6 regulates NKT cell function are largely unknown. To investigate the effect of Slamf6 on peripheral NKT cell populations, we challenged C57BL/6 (B6) or B6.Slamf6−/− mice with the NKT-specific agonist αGalCer. Examination of liver NKT cell numbers 3 days after challenge revealed a 50-fold increase in B6 mice over vehicle-treated controls. In contrast, we observed a 270-fold increase in NKT cells in B6.Slamf6−/− mice versus controls. An analysis of spleen NKT cells yielded similar results. A comparison of in vivo BrdU uptake by NKT cells between B6 and B6.Slamf6−/− mice revealed no significant differences in proliferation. In addition, NKT cell intracellular IFN-γ, IL-4, and TNF production between B6 and B6.Slamf6−/− mice after administration of αGalCer revealed no difference in the production of these cytokines. We next evaluated the Slamf6-specific effect on NKT cell expansion using an in vitro co-culture system. Cross-linking of Slamf6 on purified B6 NKT cells resulted in diminished NKT cell expansion and increased numbers of TUNEL+ NKT cells. Taken together, these data support a model where Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist by regulating the death threshold of NKT cells. These data suggest that Slamf6 blockade could be a useful tool to manipulate the expansion of NKT cells in vivo.

Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells.

BackgroundPluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.MethodsVSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.ResultsNuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN–/CD45– VSELs and slightly larger LIN–/CD45+ HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.ConclusionBoth VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0311-6) contains supplementary material, which is available to authorized users.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

The expression of IGF-1R in Helicobacter pylori-infected intestinal metaplasia and gastric cancer.

Overexpression of IGF-1R has been demonstrated in gastrointestinal cancers, and its expression is reported as the result of the loss of tumor suppressors. IL-16 is involved in the pathophysiological process of chronic inflammatory diseases. The aim of this study is to determine the changes in the expression of IGF-1R in intestinal metaplasia (IM) and gastric cancer (GC) as well as the effect of Helicobacter pylori (H. pylori) and IL-16 on cell proliferation and IGF-1R expression in gastric cells. AGS cells were incubated with combinations of IL-16 and H. pylori. Gastric cell proliferation was studied by BrdU uptake. In H. pylori infected mucosa, IGF-1R was significantly higher in IM than chronic gastritis (CG), and also higher in GC than CG and IM. H. pylori significantly decreased BrdU uptake. IL-16 increased BrdU uptake and IGF-1R on AGS cells which had been decreased by H. pylori. Co-incubation with IL-16 increased the expression of IGF-1R mRNA in H. pylori infected cells. We conclude that the expression of IGF-1R in H. pylori infected gastric mucosa may indicate an early stage of carcinogenesis. The IL-16 secretion by H. pylori can be a trigger for the expression of IGF-1R, and it may also be a factor for gastric carcinogenesis.

Cytotoxic Effect of 5-Bromo-2-Deoxyuridine on Olfactory Epithelium

In vertebrates the most notable feature of the olfactory epithelium (OE) is its ability to continually replace cell types, including mature sensory neurons. In amphibians, the neuronal turnover is observed in adult animals and during larval development also. Today, one of the most used techniques for studies of cell proliferation and differentiation is the 5-bromo-2-deoxyuridine (BrdU) uptake, a synthetic analogue of thymidine that is incorporated into cells during DNA replication. Despite this, and because of its toxicity, some authors have questioned its use for the above mentioned purposes. This work analyses BrdU incorporation technique for cell proliferation and differentiation studies within the OE of amphibian larvae. The methodology used has been validated in Xenopus laevis larvae for studies related to inner ear formation. However, we observe a large number of adverse effects on cellular components of the OE, such as an increase in apoptosis, alterations in the expression pattern of different cell markers, as well as defects in the foraging behavior in animals that were exposed to BrdU. We conclude that even though BrdU can be successfully used as a proliferation cellular marker in the OE, It should be very careful when BrdU is used for studying cell lineage.

Abstract 12739: Microrna-294 Modulates Cardiomyocyte Proliferation Following Myocardial Infarction

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction. Methods and Results: miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages while the expression was lost in the neonates and adults as confirmed by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2, E1, CDK2 together with c-myc, E2F1 and E2F3 was observed in NRVMs treated with 25nM mimic for miR-294. Additionally, miR-294 treated NRVMs showed in AKT phosphorylation along with enhanced protein levels of cyclin D1 and E2F1. Increased expression of p-histone 3, Ki67 and Aurora B kinase (G2/M) was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Administration of miR-294 in mice subjected to myocardial infarction demonstrated augmentation of cardiac function in mice receiving miR-294 8 weeks after injury. Increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, p-Histone 3 and Aurora B expression by immunostaining. Concurrently, a decrease in infarct size along with decreased apoptosis was observed in the miR-294 hearts compared to the control. Conclusion: Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.

Abstract 13212: The Role of Macrophage STAT3 Signaling in Pathogenesis of Aortic Dissection

Aortic dissection (AD) is a fatal disease caused by the disruption of the intimomedial layer. Recent studies showed that IL-6, a STAT3-activating cytokine, and macrophages play important roles in AD. However, it is unknown whether or how activation of macrophage STAT3 is involved in AD pathogenesis. To answer this question, we examined both mouse AD model and human AD tissue. We used macrophage-specific knockout of SOCS3 (mSOCS3-KO), a negative regulator of STAT3, to specifically sensitize macrophages to STAT3 activation, and compared them with wild type (WT) mice. We created a mouse model of aortic stiffening by periaortic CaCl2 treatment of abdominal aorta, and infused them with angiotensin II (Ca+AngII), which augmented hemodynamic stress to aortic walls. Both WT and mSOCS3-KO showed microscopic injuries in aorta with equal frequencies (40%) 1 week after Ca+AngII. Whereas WT showed fibrotic repair of the injury, mSOCS3-KO failed to show such reparative response and developed large AD in 6 weeks. We analyzed the aorta 1 week after the Ca+AngII stimulation, focusing on the changes in aorta preceding AD development. Transcriptome analysis showed higher activation of cell cycle followed by higher proinflammatory response in mSOCS3-KO than in WT aorta before AD development. Ki67 staining and BrdU uptake study confirmed the proliferative response of inflammatory cells in aorta. Flowcytometric analysis showed preferential differentiation of mSOCS3-KO macrophages to proinflammatory M1 with reduction in reparative M2 population (M1:M2 = 2:1), while WT macrophages differentiated equally to M1 and M2. Immunofluorescence study of human AD tissue showed the infiltration of CD68-positive macrophages in adventitia and outer media at the site of dissection and surrounding intact aortic walls. The infiltrating cells showed high phospho- (active) STAT3 and Ki67. Interestingly, STAT3 was less active in tissue with fibrotic changes, suggesting the negative association of STAT3 activation and tissue repair. Therefore, macrophage STAT3 activation resulted in higher proliferation and M1 differentiation, which is likely to promote AD development. Over activation of STAT3 in macrophages may be a therapeutic target to mitigate the destructive process in AD.

BE360, a new selective estrogen receptor modulator, produces antidepressant and antidementia effects through the enhancement of hippocampal cell proliferation in olfactory bulbectomized mice

We have reported that the carborane compound BE360 is a novel selective estrogen receptor modulator and new therapy option for osteoporosis. The aim of this study was to explore the effects and underlying mechanisms of BE360 on depressive-like behavior and memory impairment in the olfactory bulbectomized (OBX) mice, an experimental animal model of depression and dementia. BE360 was administered subcutaneously to mice using a mini-osmotic pump for 2 weeks. Depressive-like behavior was measured as the reduced intake of a sweet solution in the sucrose preference test. Short-term memory was assessed using the Y-maze test. Cell proliferation was assessed by the analysis of cells expressing 5-bromo-2′-deoxyuridine (BrdU) uptake. The expression of phosphorylated cyclic-AMP response element binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) were measured by immunoblot. The depressive-like behavior and memory impairment in OBX mice were improved by the chronic treatment with BE360. Immunohistochemical analysis showed that the number of BrdU-positive cells in the dentate gyrus of the hippocampus significantly decreased in OBX mice whereas they increased after the chronic treatment with BE360. Immunoblotting studies revealed that pCREB and BDNF were significantly increased in the hippocampus of OBX mice treated with BE360. The present study has shown that BE360 has antidepressant and antidementia effects characterized by hippocampal cell proliferation potentially activated via CREB/BDNF signaling pathways. These results indicate that BE360 may have valuable therapeutic potential against depression and neurodegenerative diseases.

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

Abstract 106: Pluripotent Stem Cell Microrna-294 Induces Cardiomyocyte Proliferation and Augments Cardiac Function After Myocardial Infarction

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction. Methods and Results: miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages while the expression was lost in the neonates and adults as confirmed by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2, E1, CDK2 together with c-myc, E2F1 and E2F3 was observed in NRVMs treated with 25nM mimic for miR-294. Additionally, miR-294 treated NRVMs showed in AKT phosphorylation along with enhanced protein levels of cyclin D1 and E2F1. Increased expression of p-histone 3, Ki67 and Aurora B kinase (G2/M) was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Administration of miR-294 in mice subjected to myocardial infarction demonstrated augmentation of cardiac function in mice receiving miR-294 8 weeks after injury. Increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, p-Histone 3 and Aurora B expression by immunostaining. Concurrently, a decrease in infarct size along with decreased apoptosis was observed in the miR-294 hearts compared to the control. Conclusion: Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.

Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging.

Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging.

0190 : Ephrin-B1 blocks adult cardiomyocyte proliferation and cardiac tissue regeneration

The possibility to reboot the proliferation of adult resident cardiomyocytes (CM) has recently emerged as a new promising avenue in cardiac regenerative medicine. For that purpose, there is an urgent need for identifying the molecular mechanisms involved in the natural blockage of adult CM proliferation. We recently identified ephrin-B1 as specific stabilizer of the CM rod-shape allowing the cardiac tissue cohesion. Interestingly, we found that ephrin-B1 knock-out mice (KO) compensate aging stress through a surprising CM hyperplasia, suggesting an atypical proliferation of adult CM. Cell cycle genes profiling performed by qRT-PCR showed that old KO CM significantly upregulated genes involved in all cell cycle phases. Progression of CM throughout the cell cycle was confirmed by flow cytometry and revealed the presence of the replicative S-phase only in old KO CM. Proliferation was next directly assessed on isolated 2 months-old CM stimulated or not with neuregulin- 1 growth factor. Remarkably, while this treatment resulted in 0.17% of BrdU uptake in WT CM, it increased up to 9.57% in KO. KO CM also exhibited significant higher mitotic (pH3+) and cytokinesis (AuroraB+) events compared to WT. Thus, the capacity for adult KO CM to proliferate is not restricted to aged CM but more likely an intrinsic potential of young KO CM. We next assessed the proliferative capacity of 2 month-old KO mice in vivo using the apectomy model. Remarkably, while WT mice developed a classical healing process (fibrosis/inflammation), KO mice almost completely regenerated the apex as indicated by the presence of mitotic (BrdU+, pH3+, AuroraB+) CM and significant reduced fibrosis (50%) in the resected zone. These results demonstrated that ephrin-B1 protein is a specific blocker of adult CM proliferation and its downregulation represents a huge interest for future therapeutic approaches in cardiac regenerative medicine.

The Potential Role of the Glycoprotein Osteoactivin/Glycoprotein Nonmetastatic Melanoma Protein B in Pancreatic Cancer

Pancreatic ductal adenocarcinoma is still one of the deadliest solid cancers so the finding of new therapeutic approaches and novel targets are of utmost importance. Glycoprotein nonmetastatic melanoma protein B (GPNMB), initially termed glycoprotein nonmetastatic gene B and also named osteoactivin (OA), is a type 1 transmembrane protein that has been recently found to play a role in cancer cell proliferation, angiogenesis, and invasion. Due to its potential responsibility in cancer aggressiveness, the main objective of this work was to assess the role of GPNMB/OA in human pancreatic cancer. Using the human pancreatic cancer cell line Panc-1 in vitro, the effects of GPNMB on growth, proliferation, and invasion were tested by BrdU uptake, cell cycle and Annexin V-FITC analysis, RT-PCR, protein expression, and invasion chamber assays. Our results showed that GPNMB/OA protein expression prevents cells from apoptosis-enhancing proliferation and represents a novel modulator of the invasion and metastasis in pancreatic cancer cells. Due to its main membrane localization in cancer cells and its role in the aggressiveness of pancreatic cancer, GPNMB/OA could represent a novel targeted therapy for pancreatic cancer being attractive for antibody-based therapies.

Effects of Kupffer cell inactivation on graft survival and liver regeneration after partial liver transplantation in rats

Gadolinium chloride (GdCl3) selectively inactivates Kupffer cells and protects against ischemia/reperfusion and endotoxin injury. However, the effect of Kupffer cell inactivation on liver regeneration after partial liver transplantation (PLTx) is not clear. This study was to investigate the role of GdCl3 pretreatment in graft function after PLTx, and to explore the potential mechanism involved in this process. PLTx (30% partial liver transplantation) was performed using Kamada's cuff technique, without hepatic artery reconstruction. Rats were randomly divided into the control low-dose (5 mg/kg) and high-dose (10 mg/kg) GdCl3 groups. Liver injury was determined by the plasma levels of alanine aminotransferase and aspartate aminotransferase, liver regeneration by PCNA staining and BrdU uptake, apoptosis by TUNEL assay. IL-6 and p-STAT3 levels were measured by ELISA and Western blotting. GdCl3 depleted Kupffer cells and decreased animal survival rates, but did not significantly affect alanine aminotransferase and aspartate aminotransferase (P>0.05). GdCl3 pretreatment induced apoptosis and inhibited IL-6 overexpression and STAT3 phosphorylation after PLTx in graft tissues. Kupffer cells may contribute to the liver regeneration after PLTx through inhibition of apoptosis and activation of the IL-6/p-STAT3 signal pathway.

An absence of nuclear lamins in keratinocytes leads to ichthyosis, defective epidermal barrier function, and intrusion of nuclear membranes and endoplasmic reticulum into the nuclear chromatin.

B-type lamins (lamins B1 and B2) have been considered to be essential for many crucial functions in the cell nucleus (e.g., DNA replication and mitotic spindle formation). However, this view has been challenged by the observation that an absence of both B-type lamins in keratinocytes had no effect on cell proliferation or the development of skin and hair. The latter findings raised the possibility that the functions of B-type lamins are subserved by lamins A and C. To explore that idea, we created mice lacking all nuclear lamins in keratinocytes. Those mice developed ichthyosis and a skin barrier defect, which led to death from dehydration within a few days after birth. Microscopy of nuclear-lamin-deficient skin revealed hyperkeratosis and a disordered stratum corneum with an accumulation of neutral lipid droplets; however, BrdU incorporation into keratinocytes was normal. Skin grafting experiments confirmed the stratum corneum abnormalities and normal BrdU uptake. Interestingly, the absence of nuclear lamins in keratinocytes resulted in an interspersion of nuclear/endoplasmic reticulum membranes with the chromatin. Thus, a key function of the nuclear lamina is to serve as a "fence" and prevent the incursion of cytoplasmic organelles into the nuclear chromatin.

Abstract 16788: Delivery of Pluripotent Stem Cell Specific Microrna-294 Induces Cardiomyocyte Proliferation Augmenting Cardiac Function After Myocardial Infarction

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction. Methods and Results: miR expression analysis was carried out in the heart during development to determine levels of miR-294. Elevated level of miR-294 was observed in the prenatal heart confirmed by qRT-PCR. In contrast, miR-294 expression is lost in the neonates and adult mice. To further assess the effect of miR-294, neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2 and E1 together with CDK2 was observed in NRVMs treated with 25nM mimic for miR-294. Increased expression of p-histone 3, PCNA (S-phase), Aurora B kinase (G2/M) and Ki67 was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Intramyocardial administration of miR-294 was carried out in mice at the time of myocardial infarction to determine whether miR-294 delivery increases cardiomyocyte proliferation and survival in response to injury. Enhance cardiac function was observed in mice receiving miR-294 3 weeks after myocardial infarction. Concurrently, increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, PCNA and Aurora B expression by immunostaining. Conclusion: Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.

Abstract 16492: A Common Gene Network Governs the Cellular Phenotypes in Acute Aortic Dissection

Acute aortic dissection (AAD) is a common disease with sudden onset and high mortality, for which pathogenesis is largely unknown. To better understand AAD pathogenesis, we examined three mouse models of AAD; knockout mice for tenascin C, an extracellular glycoprotein that is secreted by vascular smooth muscle cells (VSMCs), macrophage-specific knockout of SOCS3 (mSOCS3-KO), a negative regulator of JAK/STAT signaling, and infusion of beta-aminopropionitrile, that blocks crosslinking of elastin and collagens. All of three models develop AAD upon the continuous infusion of angiotensin II (AngII). We performed transcriptome analyses at the early time point of AngII infusion when the aortic structure showed no obvious abnormality. Interestingly, the gene expression patterns showed common changes; induction of proinflammatory molecules and cell cycle machinery, and suppression of the structural extracellular matrix (ECM) and cytoskeletal molecules of VSMCs. Such a pattern is consistent with that in human AAD as previously reported. We performed detailed analysis in mSOCS3-KO. Bayesian network model revealed that upregulation of cell cycle machinery precedes other changes in the gene expression. Cell cycle analyses using Ki67 staining and BrdU uptake assay showed that AngII caused proliferative response before AAD development both in macrophages and in VSMCs, indicating the macrophage-VSMC interaction. In addition, the proliferative response of macrophages was associated with the preferential differentiation into the proinflammatory M1 phenotype as determined by the high expression of Ly6C. Our findings showed a gene expression network of AAD that is common to human and mouse models. The findings in mouse models suggest that cell cycle activation precedes both the proinflammatory response in macrophages and the functional deterioration of VSMCs, which in turn would weaken the aortic wall strength. In addition, tissue-specific knockout studies revealed that interactions between macrophages and VSMCs underlie the gene expression network in AAD. Deciphering such cellular interactions and gene expressions would lead to better understanding of this fatal disease.

Cyclin-dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice

Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin-dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild-type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5-bromo-2′-deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion.