Furthermore, patients with combined reduction of PCAF and PACS1 had significantly poorer overall survival (p = 0.0257), confirmed in an independent dataset of 359 patients (p = 5.8 × 10e-6). At the protein level, PCAF, ADA3, and PACS1 expression were all significantly down-regulated in intestinal-type gastric cancer, and correlated with reduced progression free survival. We conclude that a pro-apoptotic mechanism centered on the intrinsic  pathway and regulated by PCAF/ADA3 can influence the progression from premalignant to malignant change, and thus act as a tumor suppression mechanism in gastric cancer.The loss of p300/CBP-associated protein (PCAF) expression is associated with poor clinical outcome in gastric cancer, and a potential bio-marker for invasive and aggressive tumors. However, the mechanism linking loss of PCAF to the onset of gastric cancer has not been identified. Given that PCAF and its binding partner transcriptional adaptor protein 3 (ADA3) were recently shown to regulate the intrinsic  pathway to apoptosis via epigenetic regulation of phosphofurin acidic cluster sorting proteins 1 and 2 , we analyzed PCAF, ADA3, and PACS1/2 expression in 99 patient-matched surgical samples ranging from normal gastric mucosa, through pre-malignant chronic gastritis and intestinal metaplasia to stage I–III invasive cancers. PCAF mRNA levels were not reduced in either pre-malignant state but were significantly down-regulated in all stages of gastric cancer, commencing at AJCC stage I ( For example, PCAF can acetylate p53 in response to DNA damage resulting in cell cycle arrest2, or the p65 subunit of NF-κB3, an important regulator of genes associated with inflammation and immunity. The significance of PCAF as a tumor suppressor is highlighted by recent studies demonstrating loss of PCAF in esophageal, breast, ovarian, colorectal, and pancreatic cancers8. In esophageal squamous cell carcinoma, restoring PCAF expression by reversing hypermethylation of the PCAF promoter was associated with suppression of tumor cell growth8.The epigenetic modifier p300/CBP-associated factor (PCAF) is a histone acetyl transferase (HAT), the silencing of which is associated with a number of cancers. PCAF is a key component of various heterotypic protein complexes that regulate multiple tumor suppressors and oncogenes, as well as transcription factors that govern cell cycle progression and cell differentiation9, the fifth most frequent malignancy and third commonest cause of cancer deaths, globally11. Stomach cancer is often detected when the disease is well advanced and curative surgical resection is no longer possible, so that fewer than 20% of patients remain alive 5 years beyond diagnosis. The disease is classified histologically by the Lauren classification into intestinal, diffuse or mixed subtypes12. While our understanding of the pathogenesis of gastric cancer remains incomplete13, there is compelling evidence that chronic gastritis (CG) arising from Helicobacter pylori infection is followed by intestinal metaplasia (IM), a key precursor of malignant change14. In their study, Ying and colleagues9, found that reduction in PCAF protein expression was associated with reduced disease free interval, although overall survival was unaffected. The authors also showed that reconstituting PCAF expression in an in vitro gastric cancer cell line reduced both its clonogenicity in soft agar and the growth of tumor xenografts in immunosuppressed mice.PCAF expression is also commonly reduced in stomach cancer15. To date, PCAF has been known as a direct regulator of cell growth and proliferation.  However, our screen independently identified PCAF and its binding partner human transcriptional adaptor 3 (ADA3) as proteins whose down-regulation rendered cancer cells refractory to granzyme B delivered by the pore-forming protein perforin15. ADA3 is also known to be a critical regulator of DNA repair, and its mislocalization in cells is linked to adverse outcomes in human breast cancer16.We used short hairpin (shRNA) technology to knockdown expression of >1200 candidate genes involved in cell death signaling to identify those that specifically regulate apoptosis activated by the human pro-apoptotic protease granzyme B. This key immune effector protease is secreted by cytotoxic T cells and natural killer cells and brings about target cell death through the mitochondrial pathwayPACS1 and PACS2 genes are regulated epigenetically by PCAF and ADA3, acting in concert17. Consequently, knockdown of either PACS protein resulted in survival of cervical cancer and colon cancer cells in vitro, phenocopying the loss of PCAF or ADA3 expression17. We found that loss of PACS1 or PACS2 protected cells against stimuli that induce mitochondrial outer membrane permeabilisation (MOMP), but through different mechanisms17. The PACS protein family regulates protein sorting and trafficking through generic processes including the endosomal transport system18, and we showed that disrupting these trafficking pathways suppresses MOMP. In the case of PACS2, this occurs by reducing the activation of the upstream apoptotic mediator BH3 interacting-death domain agonist (BID)15, the key substrate cleaved by human granzyme B in the target cell cytosol21. The reduced generation of activated (truncated) Bid resulted in reduced MOMP, and improved cell survival15. By contrast, loss of PACS1 had no effect on Bid trafficking or cleavage; rather, knockdown of PACS1 resulted in disturbed BAX/BAK oligomerization in response to truncated BID and to other BH3-only cell death agonists17. Consequently, loss of PACS1 resulted in protection against a broader array of apoptotic stimuli operating through MOMP, including UV radiation and drugs such as staurosporine and etoposide, as well as to granzyme B17.Our studies also further elucidated the tumor-suppressing functions of PCAF and ADA3: mechanistically, we showed that enhanced cancer cell survival in response to reduced PCAF or ADA3 was related to reduced expression of phosphofurin acidic cluster sorting proteins 1 and 2 , as both the 9 did not examine expression of ADA3, PACS1, or PACS2, we decided to determine whether PCAF and/or other components of the apoptotic pathway are down-regulated in primary human gastric cancer and/or in pathologies known to be precursors of malignant transformation.Taken together, the data across our previous studies indicated that PCAF, ADA3, PACS1, and PACS2 together constitute a novel pathway that facilitates cell death through the intrinsic apoptotic pathway, with PCAF and ADA3 playing crucial roles in epigenetically regulating PACS1 or PACS2, which in turn influence MOMP. Given that the previous studies linking PCAF depletion with poor outcomes in stomach and several other forms of cancer9. To explore this further, we interrogated samples collected from a cohort of 99 patients collected at the time of surgical resection, and which had previously been profiled for their mRNA expression22. For some of these patients (n = 45), adjacent normal and pre-malignant tissue was also collected and profiled. Histological analysis classified these as normal gastric mucosa (NM), chronic gastritis , or intestinal metaplasia . Table A previous study identified a significant reduction in PCAF protein expression in human intestinal-type stomach cancer, however mRNA levels were not reportedp < 0.01). When the data were further analyzed to take into account stage of disease, the reduction in PCAF mRNA was evident across all stages for intestinal-type cancers, including the earliest stage, AJCC stage I . A similar trend was noted for the diffuse tumors, however the difference only became statistically significant for later stage cancers. Mean PCAF mRNA expression levels were not reduced in stage 3 disease compared with stage I , suggesting that PCAF expression is lost early in the development of gastric cancer, possibly in the transition from pre-malignant IM to invasive cancer . We then further compared PCAF expression levels in paired samples from individual patients, and found that mRNA levels were invariably reduced in the cancer, in comparison with premalignant lesions from the same patient .Given that the previous study (9) examined only protein expression, we first quantified the relative PCAF mRNA levels across the cohort. When we pooled the data across the cohort, PCAF expression levels were significantly reduced in both intestinal-type and diffuse cancer tissue, compared to CG and IM . A similar reduction in PACS1 mRNA was observed in intestinal-type cancer tissue compared to IM . A similar trend was noted for diffuse cancers but failed to reach statistical significance . The observation was validated using a larger, independent dataset comprising 359 gastric cancer patients23, confirming the significant role of this pathway in gastric carcinogenesis . There was significantly shorter progression free survival (PFS) for patients with low mRNA expression of either PCAF or PACS2 . A similar trend was noted for both PACS1 and ADA3 mRNA levels, but neither reached statistical significance  correlated with clinical outcome. We found no difference in overall survival when the expression levels for PCAF, ADA3, PACS1, or PACS2 mRNA were considered singly (data not shown). However, patients who had reduced expression of both PCAF and PACS1 had significantly reduced overall survival , and an even stronger association when the percentage stained area was compared for matched malignant and adjacent normal mucosa from the same individual .Given that the pro-apoptotic proteins PCAF, ADA3, and PACS1 were significantly down regulated in tumor compared to premalignant gastric mucosa at the mRNA level, we examined the expression of the corresponding proteins by immunohistochemistry in a tissue microarray (TMA). The TMA comprised 73 resected stomach cancer specimens and 70 specimens of adjacent non-malignant and premalignant gastric mucosa from the same patients Table . Stainin24. The specific mechanisms of tumorigenesis that lead to gastric cancer are incompletely understood. It has previously been observed that the HAT protein PCAF is significantly reduced in human gastric cancer tissue, particularly the intestinal sub-type25. For this reason, we examined tumor, and where possible, matched normal mucosa and pre-malignant tissues  from a cohort of gastric cancer patients diagnosed with intestinal, diffuse or mixed gastric tumors. We became interested in the clinical observations on PCAF expression because we had independently found from performing a functional genomics (shRNA knock-down) screen that loss of PCAF or its binding partner ADA3 resulted in protection of cancer cell lines from a range of apoptotic stimuli such as anti-cancer drugs, UV irradiation and human granzyme B, that operate by inducing MOMP17. The studies were carefully controlled and were performed in an isogenic setting; that is, we used either sh or siRNA knock down of individual mRNAs in the same cells and compared the effects to scrambled or irrelevant targeting constructs. We also used immunoblotting and quantitative PCR to ensure that our cellular manipulations had no effect on the expression levels of a broad range of other proteins that regulate MOMP17. Our study design was also influenced by more recent advances in our understanding of how PCAF and ADA3 epigenetically co-regulate MOMP to bring about apoptotic cell death, and the involvement of downstream PCAF/ADA3 targets PACS1 and PACS2 in that process17. Using similar isogenic settings, we showed that the loss of expression of PACS1/2 phenocopied the loss of ADA3/PCAF, which epigenetically regulate their expression.Gastric cancer is a deadly disease, a result of asymptomatic early disease leading to late diagnosis and a lack of effective treatment options26. PCAF and ADA3 are critically implicated in cell death mediated through the intrinsic pathway and directly regulate PACS1 and PACS2 expression17. PACS1 is an adaptor protein that shuttles other proteins between the trans Golgi network and endosomes. It typically binds to proteins with acidic cluster motifs and its cargoes thus include a broad variety of proteases, receptors, ion channels and viral proteins, implicating PACS1 as an important factor in maintaining cellular homeostasis27. The specific function of PACS1 in apoptosis has not been completely deciphered, but its absence results in the formation of aberrant Bax/Bak multimers and reduced MOMP in cells exposed to a variety of death stimuli17. Mutation or deletions of the PACS1 gene locus at chromosome 11q13.1 has been associated with the development of several cancers including cervical cancer29. Importantly, we have also observed that independently reducing PACS1 expression levels endows significant protection against cell death stimuli that operate through the mitochondrial pathway, phenocopying the loss of PCAF/ADA317. The loss of PACS2 did not qualitatively affect the formation of Bax/Bak oligomers, but exerted a similar effect on MOMP by reducing the processing (or possibly the trafficking) of the BH3-only protein BID, thus damping the death signaling pathways that culminate in Bax/Bak oligomerisation15.Along with its binding partner ADA3, PCAF resides in a multi-protein complex that regulates gene transcription23 adding considerable weight to our findings  , but remained at normal levels in adjacent non-malignant or pre-malignant tissues, including in a large series of matched controls. Similar changes were also observed for ADA3 and PACS1. The changes were common to both the intestinal and diffuse forms of the disease: PCAF was significantly reduced in diffuse gastric cancer, PACS1 in the intestinal form, and ADA3 in both subtypes. Overall survival was significantly reduced for patients where the expression of both PACS1 and PCAF were diminished. The latter observation was confirmed in a larger, independent cohort of gastric cancer patients17. Our report is the first to link reduced PACS1 levels with gastric cancer. The recently demonstrated pro-apoptotic properties of PACS1 and PACS2 and their transcriptional regulation by ADA3 and PCAF suggests that the four proteins (and possibly others) constitute a novel signaling pathway that acts to suppress the formation of gastric (and possibly some other) cancers. The fact that these changes were not associated with either CG or IM provides early evidence for the hypothesis that defects of this pathway may prompt the change from a pre-malignant state to invasive cancer.Reduced PCAF, ADA3, and PACS1 expression may have implications for gastric cancer tumorigenesis given our findings that PCAF and ADA3 expression are reduced on stage I disease but not in adjacent tissues affected by pre-malignant change (CG or IM), and that PACS1/PACS2 play an important role as effectors of apoptosis through the intrinsic pathway8. These studies further highlight the tumor suppressing functions of PCAF that require further exploration. In healthy cells a dynamic equilibrium state between HATs and histone deacetylases (HDACs) exists that tightly regulates gene transcription. Tumorigenesis can disrupt this equilibrium, resulting in epigenetic reprogramming that affects gene expression enhancing cell survival or proliferation of cancer cells. Current new therapies targeting epigenetic modifications such as selective and non-selective HDAC inhibitors are showing signs of success in hematological malignancies such as leukemia and some solid tumors30. However, identifying precisely which epigenetic modifiers are altered in solid tumor models would allow more rational selection of appropriate targeted therapy either as a mono therapy or in combination with conventional agents. For example, one study found that the PCAF genotype that encodes Ser/Ser at codon 386 is found significantly more frequently in hepatocellular cancer31, while others showed that the PCAF locus is frequently disrupted in esophageal cancers32. If such mutations were to cause reduced MOMP as shown by us, sensitivity to cancer drugs operating through the mitochondrial pathway might be predicted to improve by adding a Bcl-2 inhibitor to the initial therapy.PCAF and ADA3 are two core proteins residing in histone modifying complexes for example in the human ATAC or SAGA complexes and their deregulation has been associated with solid tumor development such as breast, ovarian, colon and esophageal cancersThis study highlights the potential of using PCAF, ADA3, and PACS1 as biomarkers for early invasive gastric cancer and possibly, to predict response to cancer drugs that activate the mitochondrial cell death pathway. If so, our current study suggests that tracking the expression of mRNA/antigen levels for single proteins may be less informative than in combination, for example PCAF in concert with PACS1, as suggested by this study. Further validation of these markers in other, independent cohorts of gastric cancer patients will be required to establish their significance both in gastric cancer development and as predictors of clinical outcome.n = 99) and matching premalignant samples  were collected from patients undergoing curative resection for GC from Melbourne Australia. Fresh frozen and formalin fixed tissue was collected at the time of surgery, as described previously33. RNA was extracted and profiled using Affymetrix U133+2 chips as previously described (GSE51105)22. A second, independent sample collection collected from 359 patients23 was also interrogated in some analyses, particularly to verify or refute results from our 99-subject study cohort.Gastric tumor  and 7-AAD-FITC  and fluorescence was detected on a cytofluorograph.22. The microarray data sets are available at http://www.ncbi.nlm.nih.gov/projects/geo/ (Accession: GSE1105). Raw data were normalized using RMA with Partek® software as described previously and gene expression values, represented as normalized intensity values, were extracted33. Findings were validated using quantitative real time PCR (qPCR) on gastric cancer cell lines. Cell line RNA was used for first strand cDNA synthesis as previously described15. HPRT, PACS1, PCAF, and ADA3 genes (primers available on request) were quantified using qPCR, as previously described15.RNA was extracted from cell lines and patient tissue using the RNeasy mini Kit  according to the manufacturer’s instructions. Primary tissue mRNAs were hybridized to Affymetrix Human Genome U133 plus Genechips HG-U133 Plus 2.0  according to the manufacturer’s instructions, as previously described2O2 washed with PBS 0.05% Tween and blocked in 2.5% normal horse serum. Sections were incubated in normal Rabbit IgG , PCAF , ADA3  or PACS1  overnight at 4 °C. Sections were washed with PBS 0.05% Tween, incubated in ImmPRESS™ anti-rabbit Ig reagent  and then washed with PBS 0.05% Tween. Slides were then developed in diaminobenzidine (DAB) peroxidase substrate solution , washed in water and counterstained with hematoxylin using a Leica Autostainer  and mounted. Slides were scanned using VS120 virtual slide microscope scanner  to create images for analysis. The percentage of positive (DAB) stained area for each protein of interest  was measured using MetaMorph® image analysis software . A relative area for each tissue sample was quantitated. The percentage staining for each tissue sample was calculated by normalizing the relative staining for each protein to the respective IgG relative DAB stained area. Values were then plotted to create a histogram presented as a mean±SEM or presented as paired data comparing the percentage intensity of non-malignant tissue (NM) to the paired gastric cancer tissue (tumor) samples.GC and non-malignant tissues were ethanol or formalin fixed, paraffin embedded and used to create tissue microarrays (TMAs). Each tissue array block contained up to 60 samples. Sections of 4 mm were cut from each tissue array block, deparaffinized and dehydrated. For immunohistochemistry, slides were dewaxed and rehydrated using a Leica Auto Stainer  followed by high pressure antigen retrieval. Endogenous peroxidase was blocked in 3% Ht-test or two-way ANOVA with Bonferroni adjustment by Prism software . A p-value of 0.05 was considered significant. Kaplein–Meier curves for progression free survival (PFS) were generated using Graphpad Prism. Samples were divided into those which are low (below median) and high (above median) expressing for each gene of interest. For tissue microarray analysis data was presented in the following groups: gastric tumor tissue for Intestinal (I) or Diffuse (D), non-malignant tissue Chronic Gastritis (CG) or Intestinal Metaplasia (IM).Results are presented as mean ± S.E.M. Statistical differences were evaluated by


The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial function that has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate the pore opening. We previously found that global CypD knockout mice (KO) are protected from diet-induced glucose intolerance; however, the tissue-specific function of CypD and mPTP, particularly in the control of glucose homeostasis, has not been ascertained. To this end, we performed calcium retention capacity (CRC) assay to compare the importance of CypD in the liver versus skeletal muscle. We found that liver mitochondria are more dependent on CypD for mPTP opening than skeletal muscle mitochondria. To ascertain the tissue-specific role of CypD in metabolic homeostasis, we generated liver-specific and muscle-specific CypD knockout mice  and fed them either a chow diet or 45% high-fat diet (HFD) for 14 weeks. MKO mice displayed similar body weight gain and glucose intolerance compared with wild type littermates (WT), whereas LKO mice developed greater visceral obesity, glucose intolerance and pyruvate intolerance compared with WT mice. These findings demonstrate that loss of muscle CypD is not sufficient to alter whole body glucose metabolism, while the loss of liver CypD exacerbates obesity and whole-body metabolic dysfunction in mice fed HFD. OF1 ATP synthase was reported to dimerize and form channels with behavior indistinguishable from that of the mPTP [Impaired mitochondrial function due to increased damage and/or impaired maintenance is associated with a plethora of disease conditions affecting multiple organ systems. The contribution of mitochondrial dysfunction to obesity and type 2 diabetes is particularly important since these two related disease conditions are reaching epidemic levels and are primarily attributed to poor diet and sedentary lifestyle. A key regulator of mitochondrial health is the mitochondrial permeability transition pore (mPTP). The mPTP presumably spans both the outer and inner mitochondrial membranes and when open allows the passage of molecules up to 1.5 kDa in size . Howeverthe mPTP –8.Opening of the mPTP can be triggered by a variety of events including calcium overload and exacerbated reactive oxygen species production –11. TranOF1 ATP synthase [The matrix peptidyl-prolyl cis-trans isomerase, cyclophilin D (CypD) is a critical regulator of the mPTP and directly binds to pore constituents, including ANT  and FOF1synthase  to facilsynthase –22. Thersynthase –27. In osynthase . Howeversynthase . Yet, thsynthase . Anothersynthase , with otsynthase . Two othsynthase , and hepIn an attempt to understand and dissect these inconsistencies, we assessed calcium loading-induced mPTP in mitochondria isolated from skeletal muscle and liver from global CypD KO mice and compared with those from wild type littermates. We also generated mice with either muscle-specific or liver-specific deletion of CypD  to determine their glucose tolerance when fed normal chow or 45% HFD. Our findings suggest that CypD deletion specifically in skeletal muscle has minimal metabolic consequences, whereas hepatic-specific deletion of the CypD gene led to exacerbated impairment of whole body glucose homeostasis in mice fed HFD.ad libitum and housed in 12/12 light/dark cycle at 68–72°F. Whole body CypD KO mice were obtained from Prof. Jeffrey Molkentin [f/f) mice (The Jackson Laboratory); Liver specific KO (LKO) was generated by crossing albumin-Cre mice (The Jackson Laboratory) with CypDf/f mice. The high-fat diet  was purchased from Research Diets (D12451) and fed to mice at 8 weeks of age for up to 14 weeks.All procedures were approved by the Animal Care and Use Committee at the University of Virginia. Mice were provided food and water olkentin . Skeleta2PO4, 5mM MgCl2, 1 mM EGTA and 0.2% BSA, pH 7.4). Nagarse (5 mg/ml) was added before homogenization and incubated on ice for exactly 1 min. Samples were homogenized on ice at the lowest speed for 3 x 15 sec with a 10 sec break in between. Fifteen ml of isolation buffer was added, and samples centrifuged at 700 x g for 10 min at 4°C. The supernatant was then centrifuged at 10,000 x g for 10 min at 4°C. The pellet was resuspended in 15 ml of suspension buffer  and centrifuged at 8,000 x g for 10 min at 4°C. The mitochondrial pellet was resuspended in 50 μl of suspension buffer and kept on ice. A protein assay was performed to determine protein concentration. In a 96 well plate isolated mitochondria (15 μg) were suspended into a total volume of 100 μl using mitochondrial challenge buffer  with the addition of 50 mM of Na-succinate (Sigma S2378) and 10 μM rotenone (Sigma R8875). One μl of Calcium Green 5N  was added, and incubated at room temperature for 5 min. Measurements were then started with calcium pulse (83 nmol/mg protein) every 2 min until a plateau of fluorescence was observed. The fluorescence was measured using an excitation/emission wavelength of 506/532 nm, respectively, in a FLUOstar omega plate reader (BMG Labtech). We performed quantification of calcium retention capacity by counting the number of calcium additions required to induce opening of the mPTP, observed by a sharp increase in Calcium Green fluorescence following calcium addition  fed HFD for 10 weeks as previously described , 28–29. Glucose tolerance test was performed following 6 hours fasting as described previously  with i.pAt the time of tissue harvest mice were anesthetized using 3% isoflurane and maintained under deep anesthesia with 2% isoflurane. The plantaris muscle was carefully dissected from the left leg and homogenized in western blot sample buffer. Insulin (5 U/kg) was injected i.p., and 10 minutes later the contralateral plantaris muscle was dissected and immediately homogenized in sample buffer. Total and phosphorylated Akt (Ser473) were measured by western blot as described below.Tissues homogenates were prepared as previously described . Thirty Fresh liver tissue was frozen in optimal cutting temperature compound (OCT) chilled in freezing isopentane. Five μm sections were cut using a cryostat and fixed in 10% buffered neutral formalin. Slides were rinsed in distilled water followed by a 2 min-incubation in absolute propylene glycol and a 16 hr-incubation in Oil Red O solution. Slides were then incubated in 85% propylene glycol for 1 min, rinsed in distilled water and stained in Mayer’s hematoxylin for 15–60 sec. Glass cover slips were mounted in aqueous mounting medium, and 10 images per slide were acquired using a bright field microscope. Oil Red O staining was quantified using ImageJ to assess lipid droplet size as previously described .P < 0.05 was considered statistically significant.Data are expressed as mean ± standard error of the mean. Repeated measures ANOVA was used to determine statistically significant differences between genotype during GTT and PTT. Two-way ANOVA or two-tailed Student’s t-test were used as appropriate to determine statistically significant differences between genotype and diet. 2+  were required to trigger Ca2+ overload-induced mPTP opening compared to WT mitochondria  assays in mitochondria isolated from liver or gastrocnemius muscle of WT and CypD KO mice following 10 wk of HFD. In skeletal muscle mitochondria, loss of CypD had a very minimal impact on CRC requiring only 1 to 2 more additions of Ca2+ . Loss ofchondria . These df/f mice, respectively. Muscle-specific and liver-specific CypD deletion was confirmed by immunoblot in liver, skeletal muscle, heart and fat of MKO mice  and liver-specific (LKO) CypD knockout mice by crossing muscle creatine kinase (MCK)-Cre mice or liver albumin-Cre mice with CypDMKO mice  and LKO MKO mice . DeletioMKO mice .Deletion of the CypD gene in muscle had no impact on body weight or weight gain following 11 weeks of HFD . Nor didLKO mice had similar body weight under normal chow conditions and following HFD compared with WT mice . LKO micOur objective was to dissect the tissue specific role of CypD in the control of mPTP and metabolism in the liver and skeletal muscle. The vastly enhanced calcium retention capacity in isolated mitochondria from KO liver compared with WT liver after HFD suggests that CypD plays a critical role in the regulation of mPTP opening and hence mitochondrial function in the liver. The fact that liver-specific CypD knockout mice had exacerbated glucose intolerance suggests that normal mPTP in hepatocytes is critical for them to deal with lipid overload and maintain metabolic homeostasis under the condition of HFD. Surprisingly, calcium retention capacity was only marginally enhanced in KO skeletal muscle compared with WT skeletal muscle after HFD. Although CRC in WT mitochondria was similar between liver and skeletal muscle, we observed a considerable difference between liver and skeletal muscle mitochondria when the CypD gene was deleted, and the findings are suggestive of a tissue-specific role of CypD in mPTP regulation in response to calcium overload. Although our western blot data in WT mice suggests that CypD is much more abundant in liver compared with skeletal muscle, it is important to note that this analysis was performed on whole tissue lysates with equal protein loading and the majority of skeletal muscle protein consists of structural elements. Therefore differences in mitochondrial abundance between tissues may account for the difference in CypD abundance, and we cannot use these observations to explain those from the CRC assay, which was performed with equal amounts of isolated mitochondria. Importantly, these findings paved the way for us to use molecular genetic intervention to ascertain tissue-specific function of CypD in diet-induced obesity and metabolic syndrome in mice.in vivo in a tissue specific manner. When the CypD gene was specifically deleted in muscle we observed no impact on whole body glucose tolerance, or on skeletal muscle insulin signaling assessed by Akt phosphorylation. These findings are perhaps not surprising considering the marginal impact of CypD gene deletion on isolated mitochondrial CRC from skeletal muscle. In fact, recently reported findings from Shang and colleagues (2016) showed that transient opening of the mPTP, causing ‘mitoflashes’, also displayed tissue-specific CypD dependency. They showed that frequency of mitoflashes was dramatically reduced in heart mitochondria lacking CypD, but was unaffected in skeletal muscle upon CypD deletion [We generated mice with tissue-specific deletion of the CypD gene in the liver or muscle with the goal to determine CypD/mPTP contribution to metabolic homeostasis deletion . These fIn contrast to our findings in skeletal muscle, liver-specific deletion of the CypD gene resulted in exacerbated glucose intolerance and exaggerated hepatic glucose production under the condition of HFD. The impaired whole body glucose metabolism, particularly the elevated blood glucose in LKO mice may promote storage in fat tissue and account for the increased epididymal fat deposition that we observed in these mice. The increased fat deposition may in turn contribute to the impairment in whole body glucose tolerance in LKO mice. Furthermore, although the increase in blood glucose during the PTT in LKO mice is suggestive of a liver-specific phenotype, we cannot rule out a contribution of skeletal muscle to the metabolic phenotype since we did not assess skeletal muscle insulin sensitivity in these mice. Interestingly, a recent study reported that pyruvate administration significantly increased the frequency of transient mPTP opening in cardiomyocytes, which was 42% lower in CypD KO cells . AlthougThe data presented in this study shows that CypD is a regulator of mPTP in the liver and is critical for whole body metabolic health under HFD conditions. It points to the importance of normal sensitivity to stimuli for mPTP opening in the maintenance of metabolic homeostasis in the liver under the condition of lipid overload, such as diet-induced obesity. Our previous findings of improved glucose tolerance in whole body CypD KO mice following chronic HFD  may be aS1 Fig(A) Representative Western blots of liver samples from HFD-fed CypD LKO and WT mice probed with the MitoProfile Total OXPHOS Rodent WB Antibody Cocktail to measure expression of ATP Synthase  and ETC complexes I (NDUFB8 subunit), II (SDHB subunit), III (UQCRC2 subunit) and IV (MTCO1 subunit). Expression of 14-3-3 was used as a loading control. M is 10ug of rat heart mitochondria as a control for OXPHOS protein subunits. (B) Quantification of liver OXPHOS subunit expression normalized to 14-3-3; results from three independent blots with n = 4 WT and 6 CypD LKO.(PDF)Click here for additional data file.S1 Appendix(XLSX)Click here for additional data file.


NOTCH1. In this study we tested the hypothesis that Notch-dependent and related TGF-β and BMP differentiation pathways are differently altered in aortic SMC of BAV- vs. TAV-associated aortic aneurysms. SMC were isolated from aortic tissues of the patients with BAV- or TAV-associated aortic aneurysms and from healthy donors used as controls. Gene expression was verified by qPCR and Western blotting. For TGF-β induced differentiation SMC were treated with the medium containing TGF-β1. To induce proosteogenic signaling we cultured SMC in the presence of specific osteogenic factors. Notch-dependent differentiation was induced via lentiviral transduction of SMC with activated Notch1 domain. MYOCD expression, a master gene of SMC differentiation, was down regulated in SMC of both BAV and TAV patients. Discriminant analysis of gene expression patterns included a set of contractile genes specific for SMC, Notch-related genes and proosteogenic genes and revealed that control cells form a separate cluster from both BAV and TAV group, while BAV- and TAV-derived SMC are partially distinct with some overlapping. In differentiation experiments TGF-β caused similar patterns of target gene expression for BAV- and TAV derived cells while the induction was higher in the diseased cells than in control ones. Osteogenic induction caused significant change in RUNX2 expression exclusively in BAV group. Notch activation induced significant ACTA2 expression also exclusively in BAV group. We show that Notch acts synergistically with proosteogenic factors to induce ACTA2 transcription and osteogenic differentiation. In conclusion we have found differences in responsiveness of SMC to Notch and to proosteogenic induction between BAV- and TAV-associated aortic aneurysms.Cellular and molecular mechanisms of thoracic aortic aneurysm are not clear and therapeutic approaches are mostly absent. Thoracic aortic aneurysm is associated with defective differentiation of smooth muscle cells (SMC) of aortic wall. Bicuspid aortic valve (BAV) comparing to tricuspid aortic valve (TAV) significantly predisposes to a risk of thoracic aortic aneurysms. It has been suggested recently that BAV-associated aortopathies represent a separate pathology comparing to TAV-associated dilations. The only proven candidate gene that has been associated with BAV remains  Thoracic aortic aneurysm (TAA) is a life threatening condition, which is manifested by progressive enlargement of the thoracic aorta due to destructive changes in the aortic wall. Therapeutic agents that may influence the process are absent to date and the only therapeutic decision is a surgical intervention  and changes in their functionality  is an essential factor for the SMC differentiated phenotype. This transcription cofactor is both necessary and sufficient for the development and differentiation of most SMC , SM22α (TAGLN), and calponin (CNN1) , respectively. Several lines of evidence suggest that the mechanism of aneurysm development is distinct between the two patient groups  and ligands  are expressed. Activation of Notch receptors requires binding to a transmembrane ligand presented by neighbor cells. This binding enables a series of cleavage events in the receptor, resulting in the release of the intracellular region of Notch protein . NICD, the transcriptionally active form of Notch, translocates to the nucleus where it regulates a broad range of target genes  (Table n = 14). Patients with connective tissue disorders were excluded. Control aortic specimens were obtained from organ transplant donors (n = 13) and all had TAV. All tissues were sampled from the outer curvature of the thoracic aorta.Samples of the aneurysmal wall of the thoracic aorta were harvested during aortic surgery at the Almazov Federal Medical Research Center. Eleven specimens were sampled from patients with thoracic aortic aneurysm with tricuspid aortic valve (TAV) n = 1  with 1% penicillin/streptomycin, 10 mM β-glycerolphosphate, 200 μM L-ascorbic acid and 100 nM dexamethasone (Sigma). Gene expression was analyzed 5 days after the induction of differentiation.SMC were seeded in the growth medium and after they were attached to the culture plate the medium was changed to serum-free DMEM/F12 (Invitrogen) with 1% penicillin/streptomycin. After 24 h 2,5 ng/ml of human recombinant TGF-β1 (Peprotech) was added. The cells were harvested 96 h after the addition of TGF-β1 for either qPCR or Westernblotting analyses.Lentiviral packaging plasmids were a generous gift of D. Trono ; pLVTHM was modified to bear Notch1 intracellular domain of Notch1  and by qPCR with primers to Notch1 target gene Control transductions were performed using GFP-bearing lentiviral vector.In vitro calcification was determined by alkaline-phosphatase (ALP) activity, which is an early marker of osteogenic differentiation. ALP staining was performed using Sigma BCIP®/NBT kit 14 days after the initiation of differentiation. Cells were washed with PBS and incubated with alkaline-phosphatase working solution for 10-15 min at room temperature. ALP activity appeared as blue deposition and plates were photographed with digital camera.GAPDH mRNA.Total RNA was extracted from SMCs using Extract RNA reagent  according to the instructions of the manufacturer. Total RNA (1 μg) was reverse transcribed with MMLV RT kit . Real-time PCR was performed with 1 μL cDNA and SYBRGreen PCR Mastermix  in the Light Cycler system using specific forward and reverse primers for target genes. Primer sequences are available upon request. Changes in target genes expression levels were calculated using the comparative ΔΔCT method. The mRNA levels were normalized to Proteins were extracted from SMC as follows. The cells were treated with lysis buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 5 mM EDTA (Sigma) and protease inhibitor cocktail (Roche). Extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies used for Westernblotting were the following: Notch1 , SM22α , SMA , vimentin , beta-actin , pSMAD (Abcam), calponin (SantaCruz), tubulin (Sigma).Discriminant analysis was used to investigate what parameters in gene expression could divide groups of patients. Also discriminant analysis gives an opportunity to predict what parameters are mostly common for each group.p-value significance level, and F-test were performed. A discrimination level was evaluated by assessing Wilks' lambda. Significance of an identifying criterion was determined after drawing scatterplots of canonical values and calculating classification value and Mahalanobis squared distance. Discriminant function analysis was performed with Statistica 7.0 software.Linear discriminant function analysis was performed to determine which continuous variables discriminate between groups of BAV, TAV and controls. Continuous variables were qPCR gene expression data from ΔΔCT estimation. A stepwise analysis enumerating steps, Correlation analysis was performed using R software . Spearman correlation coefficient was used. The significance of correlations was evaluated by a two-tailed Mann–Whitney test.P ≤ 0.05 was considered significant.qPCR data on gene expression was analyzed using Graph Pad Prizm. Values are expressed as means ± SEM. Groups were compared using the Mann–Whitney non-parametric test. A value of MYOCD gene encoding myocardin in smooth muscle cells (SMC) derived from aortic wall of the patients with thoracic aortic aneurysm (TAA) associated with either tricuspid- or bicuspid aortic valve (TAV- and BAV- correspondingly) and healthy controls. MYOCD expression level was significantly down regulated in the SMC of both groups of the patents , TAGLN (SM22), CNN1 , and VIM (vimentin) using the Spearman correlation coefficient  and proosteogenic genes known to be involved in calcification , TAGLN , CNN1 , POSTN (2.7- and 2.4-fold vs. 1.4-fold). Although the data on MYOCD mRNA expression did not achieve statistical significance, there was a trend toward more powerful induction of its mRNA in SMC from TAV patients comparing to the control group (p = 0.055). Thus, TGF-beta induces similar pattern of responsive gene activation for BAV and TAV-derived cells, and this activation is higher in the cells of BAV and TAV patients compared to the cells of healthy donors.In order to define whether there was a difference in TGF-β responsiveness between BAV- and TAV-derived SMC and controls, we measured the expression of phenotypic markers of the contractile SMC after treatment with human recombinant TGF-β1 Figure . ACTA2, s Figure . HoweverACTA2 and TAGLN transcript levels in both groups of diseased cells, but not in control group  Figure . For conRUNX2 transcription and Notch target gene transcription HEY1 in both normal and osteogenic medium. In contrary ACTA2 transcription was significantly induced by NICD only in osteogenic conditions  and ACTA2 (smooth muscle actin) genes  in control and BAV-derived SMC Figure , but notOur data suggest that the observed “imbalance” between myocardin and smooth muscle-specific genes expression in aortic SMC from BAV and TAV-associated aneurysms is likely to depend on additional still unknown signals that can modulate a role of myocardin in directing an endogenous SMC differentiation program in aneurysm and this requires further research.TGF-β is among the most potent factors that promote and maintain the vascular SMC contractile phenotype by upregulating smooth muscle structural genes. We revealed that aortic SMC from BAV- and TAV-related aneurysms are more responsive to pro-differentiation effect of the exogenous TGF-β1 than SMC from healthy aortas. However, we did not observe any difference in the response to TGF-β1 between BAV- and TAV-derived SMC. TGF-β pathway is apparently involved in TAA progression. Accumulating evidences are associated with increased TGF-β signaling in aneurysms (Forte et al., The discriminant analysis of expression of genes responsible for SMC differentiation state and genes related to Notch and proosteogenic regulation Figure  suggesteNotch is an important regulator of SMC (Boucher et al., RUNX2 expression and more prominent calcification in BAV-derived SMC. These results suggest a dysregulation of proosteogenic pathways in the BAV-derived cells. Notch activation induced significant elevation of ACTA2 expression and corresponding SMA protein accumulation in BAV-derived cells. It has been shown previously that the cross-talk between BMP2 and Notch signaling pathways induces osteogenic differentiation of vascular SMC (Shimizu et al., ACTA2 has been shown to directly reduce the clonal potential of human mesenchymal stem cells and to guide their differentiation toward osteoblasts. Hence, SMA not only identifies osteoprogenitors in mesenchymal populations as shown by others before (Kalajzic et al., ACTA2 in response to Notch stimulation. In addition we show that simultaneous action of osteogenic medium and Notch activation leads to elevated ACTA2 expression and to an increase in osteogenic differentiation in SMC. Thus, this Notch-dependent elevation of ACTA2 might be a direct prerequisite to calcification. Synergy between attenuated Notch and proosteogenic sensitivity could cause the final predisposition to osteogenic phenotype in BAV-derived SMC.Cardiovascular calcification is gaining an increased attention nowadays as it seems to be a “default” state of various pathologies (Hilaire et al., in vitro with isolated smooth muscle cells, but in aortic wall there is a community of SMC and endothelial cells and the communication between these different type of cells now is gaining more attention to understand proper function of the whole vessel (Lilly, This study has some important limitations. First, the groups of the patients were not quite large. Second, we worked l Lilly, . NeverthEI and DK made experiments, analyzed data and wrote the manuscript. OI, VU, NG, and OM analyzed data of the patients and collected patient samples. AG and AK analyzed data. AM designed research, analyzed data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV preferentially infects activated CD4+ T cells and mutates rapidly. The classical vaccine approach aimed to generate broad immune responses to full HIV proteins largely failed to address the potential adverse impact of increased number of activated CD4+ T cells as viral targets. Learning from natural immunity observed in a group of HIV resistant Kenyan female sex workers, we are testing a novel vaccine approach. It focuses immune response to the highly conserved sequences surrounding the HIV protease cleavage sites (PCS) to disrupt viral maturation, while limiting excessive immune activation. Our pilot studies using nonhuman primate SIV infection models suggest that this approach is feasible and promising. Worldwide, it is estimated that almost 37 million people are living with human immunodeficiency virus type 1 (HIV-1). In 2015 alone, around 2.1 million individuals became newly infected with HIV and 1.1 million people died from AIDS, highlighting the urgent need for an effective HIV vaccine. Six HIV vaccine candidates to date have been tested in Phase IIb clinical trials. The first two trials Vax004 and Vax003 sought to induce protection by eliciting antibody responses to gp120, but failed to protect against HIV acquisition .HIV primarily infects CD4+ T cells, a critical component of the human immune system. As a retrovirus HIV mutates rapidly, giving rise to extensive genetic diversity. These inherent characteristics underscore the challenges for developing a prophylactic vaccine. Novel approaches and ideas need to be tested to develop an effective vaccine to HIV-1.Edward Jenner developed the successful smallpox vaccine based on the natural immunity observed in milkmaids. Thus, the correlates of natural immunity to HIV-1 documented in highly exposed uninfected individuals may provide a vital clue for the development of a preventative vaccine for HIV-1. Several cohort studies have documented that there is considerable heterogeneity in susceptibility to HIV-1 infection –9. Some Studies showed that the observed natural resistance to HIV-1 infection in the Pumwani sex worker cohort was associated with several alleles of human leukocyte antigens (HLAs) and specific CD8+ and CD4+ T cell responses to HIV-1 –14. TherTwo things can be learned from our studies: (a) broad immune responses do not necessarily provide protection and may in fact promote infection; (b) narrowly targeted T cell response can be associated with protection against infection and may be an alternative strategy for an anti-HIV vaccine. Infection of CD4+ T cells is the key difference between HIV-1 and other infectious pathogens and activated CD4+ T cells are the primary targets for HIV-1, thus a narrow spectrum of epitope presentation by a protective allele appears to make sense. Theoretically, recognizing more epitopes will activate more CD8+ T cells to destroy the virally infected cells. However, it could also activate more bystander CD4+ T cells via cytokines produced by epitope-recognizing CD8+ T cells. The increased CD4+ T cell activation and recruitment to mucosal sites could increase the risk of HIV acquisition. This may explain why the B*07:02 allele, capable of recognizing a broader spectrum of Gag epitopes, was associated with rapid seroconversion. Ideally, an effective preventative vaccine to HIV-1 should be able to destroy the infecting virus or infected cells without causing excessive immune activation. The narrow and focused Gag epitope presentation by A*01:01 might provide such balance enabling the destruction of initially infected cells with minimum immune activation. Furthermore, recent studies have shown that in most cases mucosal acquisition of HIV-1 typically resulted from a single or a few founder viruses , 19. ImmThe only Gag peptide recognized by A*01:01 with relative high affinity and normal off-rate is a 9-mer peptide that covers the protease cleavage site at p17/p24  are the best animal models to evaluate candidate vaccines for human pathogens. PCS peptides delivered by recombinant vesicular stomatitis virus and nanoparticles (PCS vaccine) were tested in a pilot study as a preventative vaccine candidate using a cynomolgus macaque SIV infection model. Based on promising results from this pilot study, the vaccine is currently being validated and further characterized in a larger-scale study, in comparison with vaccines against full Gag and Env proteins.Based on the correlates of natural immunity to HIV resistance and our preliminary data from nonhuman primate study, the new vaccine strategy targeting the viral protease cleavage sites is feasible and promising. It needs to be further studied and refined towards the development of an effective HIV vaccine.


The restrictions imposed upon the pharmaceutical chemist due to the physical half lives of these radionuclides mandate not only a sensitive, but also a rapid quality assurance program for the finished formulations. The preparation of a class of fatty acids is an excellent illustrative example of the technical difficulties and chemical subtleties associated with the research effort on short-lived radiopharmaceutical diagnostic compounds.Positron Emission Tomography (PET) is an established method for probing biochemical processes in vivo and holds promise for being an established clinical tool –4, PET pBiologically, long chain fatty acids are important substrates utilized to meet the myocardial energy requirements. It is theorized that PET might provide insight into changes in the utilization of these compounds as a result of therapies or organ dysfunction caused by disease. Moreover, an added benefit of the successful synthesis of particular fatty acids is that it allows the radiopharmaceutical chemist the option of preparing other synthons, such as acid chlorides, aldehydes and alcohols.11C)-cyclopropanecarboxylic acid and (11C)-3,4-dimethoxybenzoic acid. These compounds are required as starting reagents for the preparation of (11C)-cyclofoxy [11C)-dopa [Fatty acids labelled with radio-carbon are prepared from the corresponding organomagnesium halide. Although numerous fatty acids have been prepared utilizing this synthetic approach, few detailed reports exist. For this reason we now report our results on the preparation of 1C-cyclopyclofoxy  and (11C11C)-carbon dioxide is produced in high radionuclidic and radiochemical purity by the 14N11C nuclear reaction on a nitrogen gas target using the Japan Steel Works Limited Baby Cyclotron (BC-1710). The 11CO2 is condensed from the nitrogen gas target stream on a vacuum line using a glass radiator trap cooled to −193 °C. Following complete removal of the nitrogen gas, the 11CO2 is allowed to react with freshly prepared organomagnesium halide. After 2–3 minutes, the reaction is quenched by acid hydrolysis. In the experiments involving the addition of carrier, known quantities of carbon dioxide were added to the cyclotron produced product.“No carrier added”  a coupling (Wurtz) reaction and b) a disproportionation reaction. These reactions are favored by elevated temperatures and excess halide concentration. Our experience indicates that the formation of the Grignard reagent proceeded smoothly when the alkyl bromide concentration was greater than 0.15 mol/L. The freshly prepared Grignard reagent exhibits a high chemical reactivity. Efforts to exclude oxygen, moisture and stable carbon dioxide are important to maintain the high specific activity and to minimize “side” reactions. Only freshly distilled solvents, flame-dried magnesium turnings and glassware, and an inert atmosphere are appropriate.The radio thin layer chromatography shows that several radioactive products are formed which lead to lower chemical yield of the labelled fatty acid. These products could be the result of competing reactions resulting in ketone formation or alcohol formation from impure or excess Grignard reagent. In the preparation of “no carrier added” radiopharmaceuticals side product formation is generally limited by restricting reaction times.In conclusion, the preparation of “no carrier added” carbon-11 labelled fatty acids is readily achievable utilizing organomagnesium compounds and carbon dioxide labelled with carbon-11; however, impure reagents and experimental conditions may reduce the concentration of the desired Grignard reagent. To find application in PET, the preparation of both radionuclidic and radiochemically pure compounds is of paramount concern. In the case of radiolabelled carboxylic acids, care must be exercised in determining the yield of Grignard reagent formation based solely upon assay of nonisolated intermediates.


A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology. Salivary glands (SG) are exocrine glands that secrete saliva, which provides lubrication necessary for proper speech, mastication, and food tasting and hence is of critical importance for oral health. Loss of saliva secretion due to impaired acinar cell function is commonly associated with autoimmune diseases such as Sjögren’s Syndrome, from γ-irradiation therapy used in patients with oral cancers, and developmental disorders–3. Patiein vitro cell culture based strategies, numerous studies have demonstrated that human and rodent stem/progenitor cells are able to rescue radiation-induced hyposalivation in mouse models., 30.+/K+nolayers . Taken t2+) dependent process with elevated intracellular Ca2+ levels acting as the primary secretory signal. To determine if mSGc exhibited functional characteristics of acinar cells, we examined the ability of mSGc to respond to stimulation by various agonists by measuring changes in intracellular Ca2+ release. As shown in 2+ concentrations increased immediately with a peak occurring after approximately 10 seconds. Interestingly, stimulation with the P2 nucleotide receptor agonist ATP showed similar results  on cells that were grown in SFM and SCM conditions and at different passages . In addition, the transcriptomic profile of the mSGc was also compared to similar data-sets based on RNA-seq results from cell lines of different developmental origin: NIH3T3 (fibroblasts), A20 (B lymphocytes) and Immortalized Mouse Oral Keratinocytes (IMOK) cells. The clo in mSGc 32–45]. –45. Krt1 in mSGc 46–50].–50.Krt17To determine how closely the overall gene expression profile of the mSGc resembled that of the salivary gland, we next compared RNA-seq datasets of mSGc to that of mouse salivary glands generated by our laboratory and several other organs and tissues. PairwisGiven the valuable role mouse models have served to better understand normal biological and pathological processes in the salivary gland, the generation of a mouse salivary gland cell line that will complement mouse transgenic and knockout studies, is important. While a recent report described the long-term culture of an immortalized submandibular salivary gland epithelial cell line, no studies evaluating the sphere forming abilities or functional characteristics of these cells were performed. Here we2+ release upon stimulation with both the muscarinic cholinergic agonist, carbachol (Cch) and the P2 nucleotide receptor agonist, ATP, suggesting secretory function of the 3D-spheroid structures.One interesting feature of the mSGc is that similar to the salivary gland, mSGc grown as monolayers express a number of salivary gland specific markers including basal (K5/K14), ductal (K8), and myoepithelial (Sma) cell markers, suggesting that these cells represent a heterogeneous population. Yet another distinguishing feature of the mSGc is their ability to organize into 3D-spheroid structures when grown in matrigel. Interestingly, we found that 3D-spheroid structures express salivary gland specific markers which show a distinct spatial organization similar to that of the salivary gland. More specifically, we observed expression of K5/K14 and Sma located towards the outer periphery of the lumen enriched spheres highlighting the ability of the mSGc derived spheres to form polarized structures. Moreover, we observed expression of the acinar cell markers Amy1, Mist1, Aqp5 and Nkcc1 which is in good agreement with the observed intracellular CaWhile determining the optimal growth conditions for mSGc, we noticed that there were no discernible differences in colony forming, clonogenic or sphere forming capabilities when grown in the presence (SCM) or absence (SFM) of serum. While there are several plausible explanations for the lack of serum-induced effects, we suspect one potential explanation is that our SCM contains relatively low levels of serum (2.5%). These levels are considerably lower than serum levels commonly used to grow and cultivate epithelial cells, which is typically 10%. Furthermore, it should be stressed that both the SFM and SCM utilized for our studies contain the growth factor EGF, which may be sufficient for mSGc survival and growth in our culture conditions. Alternatively, it is possible that the inherent nature of the immortalization process may have forced the cells to adapt to minimal and/or no growth factor requirements. Indeed, the ability of mSGc to be propagated both in the presence or absence of serum is also in good agreement with our findings in which the mSGc and primary salivary gland cells can be maintained in a defined serum free medium. These observations are not unique to the salivary gland cells described in this manuscript, as defined serum free medium is commonly used to grow primary oral and epidermal keratinocytes, 55Transcriptional profiling of mSGc at different passages and growth conditions and after comparison to other cell lines, revealed specific enrichment of genes associated with salivary gland secretion and calcium signaling, further highlighting the preservation of the tissue specific properties and functions of mSGc. Our findings from hierarchical clustering analysis reveals that mSGc grown in culture and the mouse salivary gland, share a conserved network of crucial genes and pathways and underscores the overall value and usefulness of our newly established immortalized mouse salivary cell line to better study salivary gland biology. With increased focus aimed at developing strategies to regenerate or repair salivary glands using stem/progenitor cells or using SG cells in tissue engineering approaches, there remains a need for a firm understanding of the underlying biological properties of the cell types being employed. Using a variety of molecular and biochemical approaches, we demonstrate that mSGc offer an additional resource to examine molecular and mechanistic aspects important for salivary gland development and salivary gland biology overall, with the long-term goal to treat diseases associated with salivary gland dysfunction.S1 FigAnalysis demonstrates that mSGc are exclusively of C57BL/6 mouse origin. Moreover, there is no human cell contamination as evidenced by a lack of human STR markers.(TIF)Click here for additional data file.S2 FigSpectral karyotyping  of metaphases from mSGc. An inverted DAPI image depicting chromosome banding is shown in the upper panel. Lower panel demonstrates SKY karyotype after chromosome classification.(TIF)Click here for additional data file.S3 FigQuantitative RT-PCR analysis demonstrating mRNA levels of various epithelial markers in mSGc grown as monolayers. Values were normalized to the housekeeping gene Hprt. Data are represented as the mean ±S.E. of three independent experiments.(TIF)Click here for additional data file.S4 FigProjection plots show the PCA coordinates for each of the mSGc samples, NIH3T3 (fibroblasts), Immortalized Mouse Oral Keratinocytes (IMOK) and A20 (B lymphocytes) cells.(TIF)Click here for additional data file.S5 FigA heatmap visualization of epithelial enriched genes in mSGc cells compared to A20, NIH3T3 and IMOK cells.(TIF)Click here for additional data file.S6 FigHierarchical clustering of mSGc and mouse tissues using averaged TPM values of the genes that make up the adult mouse salivary gland gene signature.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Natural cannabinoids have been used for centuries for their psychotropic properties, but their possible therapeutic implications in analgesia have been recently documented. The present review intended to make an analysis of the neuroanatomy and physiology of the cannabinoid system  and of its implications in neuropathic pain. There were also described the complex phenomena implicated in the generation and maintenance of neuropathic pain, by high lightening the implications of endogenous cannabinoids in this complex of painful conditions. The pharmacological analgesia test proves of cannabinoid implication in neuropathic pain was sustained by many studies presented in this paper. Therapeutic approaches using natural and synthetic cannabinoid receptor agonists were reviewed. Therapeutic perspectives in neuropathic pain might involve the development of new agents that influence the cannabinoid system. Thus, peripheral acting cannabinoid 1 receptors agonists, selective cannabinoid 2 receptor agonists and also modulators of endocannabinoids metabolism might be a way to success in the treatment of this complex entity called neuropathic pain. Cannabis sativa) are well known and documented since thousands of years, it was only in 1964 when Ganoi and Mechoulam identified ∆9 tetrahydrocannabinol, being the main psychotropic agent from this plant [1].Although psychotropic properties and some therapeutic actions of natural cannabinoids  [3].Initially, it was believed that these compounds act unspecifically, by influencing the neuronal membrane and this hypothesis seemed believable because of the high liposolubility of Δ9 tetrahydrocannabinol (THC) and related compounds. Lawrence & Gill proposed in 1975 that „it is unnecessary to invoke the existence of a specific cannabinoid receptor” and to propose that the psycho activity of cannabinoids results from a structure-dependent ability to disorder membrane lipids [4]. Afterwards, the first type of cannabinoid receptor was cloned out of a library of rat complementary DNA and was named CB1 [5]. The second type of cannabinoid receptor, CB2, was identified for the first time on a human promielocitary leukemia cells line [6]. After that, there were described ligands of cannabinoid receptors with a different structure than that of natural cannabinoids. Some of these structures were endogenous molecules and were named endocannabinoids. The most important endocannabinoid is probably anandamide, or arachidonoylethanolamide, isolated from water insoluble fractions from the pig brain. The name of anandamide came from Sanskrit, where ananda signified supreme happiness, bliss [7].But in the late eighties appeared some proves of the existence of some receptors for cannabinoids, proves appeared after using radioligands. The existence of some satiable bounding sites, stereo selective and with high affinity for cannabinoids, located on the mammals’ brain, was reported [8] had a 97% homology in the sequence of amino acids with the mouse or rat CB1 receptor. Two splice variants of the CB1 receptor (CB1a and CB1b) were identified, but they had smaller tissular diffusion than the initial variant (full length) [9].The human CB1 receptor (hCB1) cloned by Gerrard et al. (1991) [10] and desensitization after prolonged exposition to an agonist (more than 2 hours) [11].CB1 receptors are coupled with Gi/Go proteins and are serpentine receptors with the N-terminal end extracellular and the C-terminal end intracellular. The C-terminal end is implicated in the signal transmission to G proteins, controls the recirculation by internalization after ligand binding [12]. Through G protein action the activity of adenylyl-cyclase is diminished which leads to a decrease of cAMP level. Also through some G proteins action the activity of some ionic channels is modulated. CB1 receptors are positively coupled with potassium channels type A, M and inwardly rectifying potassium channels and negatively coupled with type L, N and P/Q of voltage dependent calcium channels and also with type D potassium channels. CB1 receptors could also activate phospholipase A through G proteins that contain Gα14, Gα15 and Gα16 subunits [13].The proximal segment of the C-terminal end of the receptor is implicated in bounding of G proteins .In the central nervous system, cannabinoid receptors type CB1 are found in the cerebral cortex, hippocampus, caudate-putamen, substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus and cerebellum [6] Unlike CB1 receptors, the CB2 ones didn’t seem to be coupled with Go type proteins or with ionic channels. They are coupled with intracellular signalization pathways associated to MAP kinase and to B-protein kinase [14].CB2 receptors were first identified in men in 1993 and then in rats and mice. The animal receptor had a 81-82% structural homology with the CB2 human receptors. [15]. There are some studies that did not showed the presence of CB2 receptors on the neurons in men and rats [6].CB2 receptors are found especially on the immune system cells. The presence of the CB2 receptors has been proved on the microglia cells and in the cerebellum in man [16].Another two serpentine receptors, being classified among orphan receptors because when discovered there did not exist a specific ligand to bind them, were supposed to be cannabinoid receptors. These two receptors are still named GPR55 and GPR119 .Cellular uptake of anandamide reportedly involves facilitated diffusion, although a specific transporter has yet to be cloned. Kinetics studies suggest the presence of an anandamide membrane transporter, and pharmacological studies using inhibitors of anandamide transport have supported the notion that anandamide transport inhibition has a role in modulating endocannabinoid tone. Among the most commonly employed drugs of this class is AM404 .Stereoselectivity of action, besides dose-analgesic effect relationship, blockade of agonists effects by substances found out to be antagonists are part of the proves for their action on specific receptors (identified to be CB1 and CB2) [16].GPR55 binds to and is activated by the cannabinoid ligand CP-55,940. In addition endocannabinoids, including anandamide and virodhamine, activate GTPγS binding via GPR55 with nanomolar potencies. Ligands such as cannabidiol and abnormal cannabidiol (extracted from cannabis) which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity on GPR55. Thus, GPR55, described as an orphan receptor, could be the third cannabinoid receptor. Another orphan receptor GPR 119, is activated by oleoylethanolamide, a substance close related to eicosanoids, and seems to be the forth cannabinoid receptor [18].Neuropathic or neurogenic pain means a complex of painful states which takes place in the nervous sensorial axis which connects the nervous sensorial endings with cortical areas. The most frequently this sensation comes from the sensorial peripheral nerves and from the posterior medullar horns [19].This type of sensation is determined by injuries of the nervous tissue others than those implicated in the simple nociception. This simple nociception is referring to injuries that don’t produce the morphological destruction of nociceptors. There are also borderline states in which pain has different components: one is given by lesions of the peripheral or central nervous system and one is due to the residual implication of nociceptors at the site of the initial tissular injury generating a mixt nociceptive-neuropathic pattern [20].Even if there are a great variety of causes implicated in the etiology of neuropathic pain, its symptoms are dominated by the dichotomy between positive and negative symptoms which correlates to the anatomic distribution of the damaged nerve. Negative symptoms include total or partial loss of sensibility, atrophy and muscular weakness. Positive symptoms include static mechanic allodynia, paresthesia, dysesthesia, and thermal hyperalgesia, painful sensations after the end of the stimulus, pain without stimuli (spontaneous), symptoms that generate continuous or intermittent paroxysmal pain [21]Allodynia, Latin name that signifies „another pain”, is an exaggerated answer (reaction) to stimuli that normally don’t produce pain. There are described: static mechanic allodynia – pain as an answer to light touch or pressure, dynamic mechanic allodynia – pain as an answer to „brushing” (touch with a cotton wool) [19]Hyperalgesia suppose exaggerated pain to a stimuli that normally don’t induce pain..At the cellular level, a very important role in these sensitization processes is hold by the synaptic transmission and by the translation of the peripheral signal to the nervous cells implicated in nociception .Areas of demyelination in otherwise normal peripheral nerves may be associated with the generation of ectopic impulses. Pain from such areas is associated with electrical-like or lancinating pain 2. Peripheral sensitization leads to changes in chemical substances from the periphery and this produces changes in spinal cord excitability [3. Pathological hyperexcitability may be secondary to changes in central control mechanisms.24]. The functional importance of Aβ-fiber sprouting is that lamina II begins to receive information about non-noxious stimuli. This information may be misinterpreted by the CNS as noxious; this is an anatomical substrate for mechanical allodynia.1. After a nerve or tissue injury, dorsal horn receptive fields may increase in size, decrease their action potential threshold and/or increase their responsiveness to nociceptive information in a way that exactly parallels changes in pain sensitivity. When peripheral nerve primary afferents are damaged, significant topographic reorganization of the primary afferent terminals in the spinal cord may occur. Thus, large myelinated mechanoceptive Aβ-axons sprout into lamina II, an area in which they do not normally terminate [2. Abnormal hyperexcitability of central nociceptive neurons appears to be highly dependent on the activation of the N-methyl-D-aspartate (NMDA) glutamatergic receptors located on the membrane of spinal cord dorsal horn neurons secondary to massive release of excitatory amino acids (particularly glutamate and aspartate).19].3. Central descending modulating systems can induce inhibition or facilitate excitability on the dorsal horn systems. Supposed opioid resistance in neuropathic pain may have several etiologies: a reduction of the inhibitory influence over spinal transmission after nerve injury as a result of decreased opioid receptors number on sensory neurons, a decrement in GABA content within the dorsal horn , and interneuron transsynaptic cell death following nerve injury. The spinal neuronal membrane is thus rendered hyperexcitable secondary to disinhibition [25].In autoradiographic studies, high affinity specific binding sites for [3H]CP-55940 have been detected not only within the spinal cord but also in rat trigeminal ganglia, pointing to the presence of cannabinoid receptors on primary afferent neurons [26].The dorsal root ganglion (DRG) has been used as a model of the peripheral nerve because of its more convenient size, location and the ability to correlate cell size and neurochemical phenotype with peripheral axon caliber. Two basic types are generally recognized in DRG: large, light A cells and small, dark B cells but some authors describe a third type: intermediate sized neurons. The largest A cells are the typical proprioceptor neurons and the small B cells are the typical nociceptor neurons [27]. Salio et al.2002 confirmed the presence of CB1Rs in small, intermediary and large cells of rat dorsal root ganglia [28].Hohmann and Herkenham (1999) showed that dorsal root ganglion cells, the source of primary afferent input to the spinal cord, synthesize cannabinoid CB1Rs. CB1R mRNA was highly expressed in dorsal root ganglion cells of heterogeneous cell size, and predominant in intermediate-sized neurons [29].Other observations indicate that under normal conditions, CB1Rs are localized mainly on non-nociceptive primary afferent fibers [30].Contrary, a study revealed that in normal rats CB1R is expressed in the majority (76–83%) of nociceptive neurons [31].The phenotypes of cells expressing CB1Rs in native DRG differs from that reported in cultured DRG, where co localization of CB1Rs with markers of nociceptors is more prevalent [32]. It is unclear if CB2Rs are expressed in satellite glial cells, the main glial cells in sensory ganglia that have been shown to be histological altered in animal models of nociception [33]. Neuronal expression of CB2R mRNA in native DRG [34] and trigeminal ganglia was similar to background under conditions in which CB1R mRNA was clearly demonstrated [35].Both CB1R and CB2R have been identified in primary cultures of dorsal root ganglion cells derived from neonatal rats [36]. This pattern might suggest an anatomical basis for the efficacy of cannabinoids in ameliorating inflammatory and neuropathic pain [37].There was considerable support for postsynaptic localization of CBRs in rat spinal dorsal horn cord [38].Presynaptic receptors were evidenced in heterogeneous dorsal root ganglion neurons and in axons of Lissauer's tract. CB1R immunoreactivity had also been localized to dorsal horn interneurons containing γ-aminobutyric acid (GABA) .Direct evidence for supraspinal sites of cannabinoid antinociception was derived from studies employing intracranial administration of cannabinoids. Site-specific injections of cannabinoid agonists to various brain regions have permitted the identification of brain loci implicated in cannabinoid antinociception. The active sites included the dorsolateral periaqueductal gray, dorsal raphe nucleus, rostroventromedial medulla, amygdala, lateral posterior and submedius regions of the thalamus, superior colliculus, and noradrenergic A5 region. These studies suggested that endocannabinoid actions at these sites were sufficient to produce antinociception [42].At the level of the periaqueductal gray, metabotropic glutamate and N-methyl-d-aspartate (NMDA) receptors were required for cannabinoid antinociception [In vitro studies demonstrated that cannabinoids inhibit GABA and glutamate release presynaptically in the periaqueductal gray in the absence of direct postsynaptic effects on periaqueductal gray neurons [43].40, 44, 45] and the nucleus reticularis gigantocellularis [44] to better characterize sites of cannabinoid-mediated antinociception. Site-specific administration of cannabinoids in the rostroventromedial medulla produced significant antinociception [40].Researchers have targeted synthetic cannabinoids at other brainstem nuclei including the rostroventromedial medulla [45].At the cellular level, it appeared that cannabinoids exert their physiological effects in the rostroventromedial medulla by presynaptic inhibition of GABAergic neurotransmission .Herzberg et al. (1997) showed that systemic application of WIN-55,212-2, a CBR agonist, was effective in reducing heat and mechanical hyperalgesia and mechanical and cold allodynia following the induction of neuropathy. These findings were obtained at doses that did not affect withdrawal latencies of hind paws contra lateral to the injury and did not produce systemic effects [provided support for a role of the endocannabinoid system in the regulation of nociceptive thresholds in hyperalgesic but not non-hyperalgesic tissue [13].Also Herzberg et al. (1997) obtained evidence that SR141716A, a selective CBR1 antagonist, enhanced thermal hyperalgesia and mechanical allodynia in rats by increasing the sensitivity to thermal and mechanical stimuli of hind paws that have been rendered hyperalgesic by unilateral sciatic nerve ligation. SR141716A did not alter the sensitivity of unlesioned paws to these stimuli. The data from this investigation the cannabinoid analgesic system might be superior to opioids in alleviating neuropathic pain syndromes [47].The same rodent neuropathy model was used to show that intrathecal injections of Δ9THC, a partial CBR agonist, were also effective in lengthening withdrawal latencies to thermal stimuli. The selective central cannabinoid receptor antagonist SR141716A, but not the generic opioid receptor antagonist naloxone, blocked the delta9-THC antinociception. It was stated that Partial ligation of the rat sciatic nerve produced similar changes to hind limb sensory stimulus thresholds like CCI, manifested as allodynia and hyperalgesia.48] showed that WIN-55,212-2 reversed the signs of neuropathy at doses that did not generally alter sensory thresholds in the contra lateral unligated limb, and there was a dose-effect relationship. This effect was prevented by co-administration of the CB1 receptor antagonist SR141716a, but not by co-administration of the CB2 receptor antagonist SR144528. Administration of SR141716a alone had no affect on the observed allodynia and hyperalgesia. These results confirm the anterior hypotheses of Herzberg et al.Bridges et al. (2001) [49]. With higher doses of these cannabinoids, anti-nociceptive effects on contralateral withdrawal thresholds and side effects of catalepsy and sedation were also observed. Of these three CBs, WIN-55,2212-2 had the best side effect profile and was also reported to reverse mechanical allodynia and reduce thermal hyperalgesia. In addition, intrathecal injections of WIN-55,212-2, as well as local injections into hind paw tissue, were effective in reducing mechanical hyperalgesia.Systemic chronic treatment with WIN-55,212-2, CP-55,940 (a complete CBR agonist) or HU210 (a complete CB1R agonist) after nerve injury produced a dose-related reversal of mechanical hyperalgesia [50]. A neural basis for reports of potent suppression by cannabinoids of the abnormal sensory responses that resulted from nerve injury was obtained.In spite of these findings, other authors showed that the effect of intravenous administration of WIN-55,212-2 appeared to be (only) centrally mediated because administration of the drug directly to the ligated nerve did not suppress the heat-evoked neuronal activity  at CCI rats [spinal nerve ligation  was dose-dependently inhibited by systemic administration of the CB2 receptor agonist AM1241 [51]. The analgesic action of AM1241 injected directly into the hind paw skin, has been demonstrated in both acute and inflammatory pain models but not so far in neuropathic animals .The role of CB2 agonists in neuropathic pain was also explored. Tactile and thermal hypersensitivity produced by spinal CB2 may be an important analgesic target [56].In vivo experiments evaluated the effects of spinal administration of JWH-133, a CB2 receptor agonist, on mechanically evoked responses of neuropathic and sham-operated rats. Spinal JWH-133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham-operated rats. Data provided evidence that at the level of the spinal cord, CB2 receptors had inhibitory effects in neuropathic, but not sham-operated rats suggesting that the activation of central, but not peripheral, cannabinoid CB2 receptors play an important role in reducing mechanical allodynia [57].Intrathecal administration of JWH133 significantly reversed partial sciatic nerve ligation-induced mechanical allodynia in mice at 0.5 h after administration. In contrast, systemic  or local  administration of JWH133 was ineffective. Furthermore, the analgesic effects of intrathecal JWH133 were absent in cannabinoid CB2 receptor knockout mice. These results suggest that 58].Intrathecal injection of CP-55940, a complete CBR agonist, was also effective in inhibiting tactile allodynia in this model, although treatment was associated with significant behavioural toxicity that could be alleviated by treatment with CB1 but not CB2 receptor antagonists [59].Costa et al. 2004 investigated the effect of repeated treatment with the synthetic cannabinoid receptor agonist WIN-55,212-2 on neuropathic pain induced in rats by chronic constriction of the sciatic nerve. WIN-55,212-2, administered daily throughout the development of neuropathy, reversed the hyperalgesia, at a dose that had no effect on the nociceptive responses of either paw contralateral to the sciatic ligation or of animals subjected to sham surgery. At 14 days after injury, the levels of mediators known to be involved in neuropathic pain, such as prostaglandin E2, nitric oxide (NO) and the neuronal nitric oxide synthetase (NOS), were increased. Repeated treatment with WIN-55,212-2 abolished these increases [Accumulating evidence suggests that cannabinoids can produce antinociception through peripheral mechanisms.60], anesthetized rats received a mild heat injury to one hind paw and exhibited hyperalgesia as evidenced by lowered withdrawal latency to radiant heat and increased withdrawal frequency to a von Frey monofilament delivered to the injured hindpaw. WIN-55,212-2 attenuated both heat and mechanical hyperalgesia dose-dependently.In another study .These data support a peripheral antihyperalgesic effect of WIN- 55,212-2 when delivered directly to the site of a nerve injury at systemically inactive doses .These data show that cannabinoids possess antihyperalgesic properties at doses that alone do not produce antinociception, and are capable of acting at both spinal and peripheral sites .The efficacy of cannabinoid receptors agonists on neuropathic pain is supported by considerable preclinical and clinical evidence and there are anecdotal reports to suggest that smoking Neural basis for reports of potent suppression by cannabinoids of the abnormal sensory responses that resulted from nerve injury was presented. Cannabinoid receptors, especially CB1 receptors, were found in the pathways of nociceptive sensations transmission and in key regions of pain control . Their stimulation produced analgesia.Studies of pain pharmacology showed a role for the endocannabinoid system in the regulation of nociceptive thresholds in hyperalgesic but not non-hyperalgesic peripheric tissues. Thus, the cannabinoid analgesic system might be superior to opioids in alleviating neuropathic pain syndromes. Recent findings showed also that spinal CB2 receptors might be an important analgesic target in neuropathic pain although they probably are not found in neurons or synapses.Accumulating evidence suggested that cannabinoids could produce antinociception through peripheral mechanisms.In order to develop new analgesic drugs that influence the cannabinoid system, drugs active in neurophatic pain and not only, it’s obvious to take into consideration 3 directions:- the development of CB1 receptors agonist drugs which do not pass the blood brain barrier. In this way, the use of liposoluble compounds active on CB1 receptors can be avoided and thus negative psychotropic effects mentioned above;- the development of CB2 receptors agonist drugs which do not have effects on CB1 receptors;- the possible use of some modulators of endocannabinoids metabolization .