Aspirin prolongs survival and reduces the number of Foxp3+ regulatory T cells in a genetically engineered mouse model of pancreatic cancer

Background and aims

Gemcitabine became the reference regimen for advanced pancreatic cancer after a randomized trial showed significant improvement in the median overall survival. Since then, the combination of gemcitabine with a variety of cytotoxic and targeted agents has generally shown no significant survival advantage as compared with gemcitabine alone. Only the addition of erlotinib to gemcitabine resulted in a significant but very small improvement in overall survival, coming along with a very uncomfortable rash in the patients. Therefore, new adjuvant agents with very low toxic levels are needed. In this study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the chemotherapeutic potential of aspirin as an adjuvant agent to gemcitabine.

Methods

Drug treatment was initiated at the age of 3 months. LsL-Kras ^G12D ; Pdx1-Cre or LsL-Kras ^G12D ; LsL-Trp53 ^R172H ; Pdx1-Cre transgenic mice were randomly assigned to receive either mock treatment, gemcitabine, or a combination of gemcitabine and aspirin. All mice were treated until death. The effect of aspirin was evaluated by histopathological analyses and immunostaining.

Results

Gemcitabine prolonged overall median survival of LsL-Kras ^G12D ; LsL-Trp53 ^R172H ; Pdx1-Cre mice by 31 days as compared to mock-treated animals (median survival, 190 vs. 159 days; p?=?0.396). Addition of aspirin to gemcitabine even extended the survival for ten more days, leading to a prolonged survival by 41 days, reaching virtually statistical significance versus the control group (median survival, 200 vs. 159 days; p?=?0.05). Furthermore, we found that administration of aspirin in combination with gemcitabine reduced the number of Foxp3⁺ regulatory T cells significantly.

Conclusion

In conclusion, we identified aspirin as an effective adjuvant agent to gemcitabine in the treatment of PDAC. While fundamental differences in biology suggest the need for caution in equating mouse tumors with their human counterparts, mouse models nevertheless represent an important source of insight regarding human neoplasia. Further studies are necessary to confirm the hypothesis that aspirin might be an effective well-tolerated chemotherapeutic adjuvant agent for pancreatic cancer.     

Presence of Pancreatic Intraepithelial Neoplasia in the Pancreatic Transection Margin does not Influence Outcome in Patients with R0 Resected Pancreatic Cancer

Background

Margin status is one of the strongest prognosticators after resection of pancreatic ductal adenocarcinoma (PDAC). The clinical significance of pancreatic intraepithelial neoplasia (PanIN) at a surgical margin has not been established.

Methods

A total of 208 patients who underwent R0 resection for PDAC between 2004 and 2008 were selected. Intraoperative frozen section slides containing the final pancreatic parenchymal transection margin were evaluated for presence or absence, number, and grade of PanINs. Data were compared to clinicopathologic factors, including patient survival.

Results

PanIN lesions were present in margins in 107 of 208 patients (51.4%). Median number of PanINs per pancreatic resection margin was 1 (range, 1–11). A total of 72 patients had PanIN-1 (34.6%), 44 had PanIN-2 (21.1%), and 16 had PanIN-3 (7.2%) at their margin. Overall median survival was 17.9 (95% confidence interval, 14–21.9) months. Neither the presence nor absence of PanIN nor histological grade had any significant correlation with important clinicopathologic characteristics. There were no significant survival differences between patients with or without PanIN lesions at the resection margin or among patients with PanIN-3 (carcinoma in situ) versus lower PanIN grades. However, patients with R1 resection had a significantly worse outcome compared with patients without invasive cancer at a margin irrespective of the presence of PanIN (P = 0.02).

Conclusions

The presence of PanINs at a resection margin does not affect survival in patients who undergo R0 resection for PDAC. These results have significant clinical implications for surgeons, because no additional resection seems to be indicated when intraoperative frozen sections reveal even high-grade PanIN lesions.     

Decreased expression of aquaporin 2 in the collecting duct of mice lacking the vasopressin V1a receptor

Background

Vasopressin V1a receptor null (V1aR^?/?) mice recently showed incomplete urinary concentration due to higher urine volume during control and water diuresis (euhydration), but showed normal response during dehydration (Aoyagi et al., Am J Physiol 295: F100–7, 2008).

Methods

Water balance, plasma vasopressin, plasma and urine osmolality, and aquaporin 2 (AQP2) expression in the kidney of wild-type (WT) and V1aR^?/? mice were therefore further examined using improved methods of urine collection (urinary bladder urine).

Results

V1aR^?/? mice demonstrated a lower urine osmolality (3,360 ± 138 vs. 3,610 ± 47 mOsm/kgH₂O) and a higher plasma osmolality (354.3 ± 1.3 vs. 342.5 ± 1.5 mOsm/kgH₂O) after dehydration for 24 h compared to WT mice (P < 0.05). In contrast, the plasma vasopressin concentration was significantly (P < 0.001) higher in the V1aR^?/? mice (48.8 ± 4.8 vs. 22.1 ± 2.4 pg/ml). On the other hand, although the AQP2 protein expression in the kidney was increased after dehydration, the basal (control) and dehydration-induced AQP2 protein levels were significantly lower in V1aR^?/? mice compared to WT mice (by Western blotting). Staining by an anti-AQP2 antibody in the luminal membrane of the collecting ducts was increased in both V1aR^?/? and WT mice after dehydration, but was relatively weaker in the V1aR^?/? mice (by immunohistochemistry). Moreover, urinary excretion of AQP2 protein, an index of the luminal AQP2 expression, was significantly (P < 0.05) lower in the V1aR^?/? mice.

Conclusion

V1aR signaling may be fundamentally important for the expression of AQP2 in the collecting ducts during control conditions and dehydration.     

RAGE Signaling Significantly Impacts Tumorigenesis and Hepatic Tumor Growth in Murine Models of Colorectal Carcinoma

Background

The receptor for advanced glycation end-products (RAGE) is a cell surface receptor implicated in tumor cell proliferation and migration. We hypothesized that RAGE signaling impacts tumorigenesis and metastatic tumor growth in murine models of colorectal carcinoma.

Materials and Methods

Tumorigenesis: Apc ^1638N/+ mice were crossed with Rage ^?/? mice in the C57BL/6 background to generate Apc ^1638N/+/Rage ^?/? mice. Metastasis: BALB/c mice underwent portal vein injection with CT26 cells (syngeneic) and received daily soluble (s)RAGE or vehicle. Rage ^?/? mice and Rage ^+/+ controls underwent portal vein injection with MC38 cells (syngeneic). Rage ^+/+ mice underwent portal vein injection with MC38 cells after stable transfection with full-length RAGE or mock transfection control.

Results

Tumorigenesis: Apc ^1638N/+/Rage ^?/? mice had reduced tumor incidence, size, and histopathologic grade. Metastasis: Pharmacological blockade of RAGE with sRAGE or genetic deletion of Rage reduced hepatic tumor incidence, nodules, and burden. Gain of function by transfection with full-length RAGE increased hepatic tumor burden compared to vector control MC38 cells.

Conclusion

RAGE signaling plays an important role in tumorigenesis and hepatic tumor growth in murine models of colorectal carcinoma. Further work is needed to target the ligand–RAGE axis for possible prophylaxis and treatment of primary and metastatic colorectal carcinoma.     

Incidentally Discovered Pancreatic Intraepithelial Neoplasia: What Is Its Clinical Significance?

Purpose

Pancreatic intraepithelial neoplasia (PanIN) is a presumed precursor of pancreatic ductal adenocarcinoma (PDAC). We assessed the relationship between incidental PanIN after resection of non-adenocarcinoma lesions and the development of metachronous PDAC in the remnant.

Methods

We retrospectively reviewed the clinicopathologic data of patients who underwent pancreatectomy for non-PDAC from January 2000 to January 2010. Intraductal papillary mucinous lesions were excluded. All available postoperative imaging and clinical follow-up data were reviewed; the risk of developing PDAC was assessed in patients with a minimum follow-up time of 6 months and with imaging studies available for review.

Results

A total of 584 patients were analyzed. Median age was 59 years (range 10–85 years), and 338 (58 %) were female. The most common lesions for which resection was performed were serous cystic neoplasms (17 %), pancreatic neuroendocrine tumors (38 %), metastatic tumors (9 %), and mucinous cystic neoplasms (7 %). PanIN was identified in 153 (26 %) patients. The majority of these patients had PanIN-1 or -2 (50 and 41 %, respectively), whereas 13 (8 %) had PanIN-3. Of the 506 (87 %) patients with adequate follow-up (median 3.7 years, range 0.5–12.6 years), 1 patient (0.2 %) with PanIN identified at the time of initial resection developed cancer in the remnant. This occurred 4.4 years after a distal pancreatectomy in the setting of PanIN-1B. No patient with PanIN-3 developed cancer during follow-up.

Conclusions

PanIN was identified in 26 % of patients who underwent resection for histopathology other than PDAC. The presence of PanIN of any grade did not result in an appreciable cancer risk in the pancreatic remnant after short-term follow-up.     

Conditional Gene Targeting in Mouse Pancreatic ��-Cells: Analysis of Ectopic Cre Transgene Expression in the Brain

OBJECTIVE

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain.

RESEARCH DESIGN AND METHODS

Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1^tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.

RESULTS

All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)^89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)^1Lphi mice were the only line that lacked Cre activity in the brain.

CONCLUSIONS

Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.In vivo analysis of gene function in the pancreas and β-cells has benefited from the development of mouse lines expressing Cre in all pancreatic compartments or restricted to the islet β-cells. The choice of promoter to drive recombinase expression is critical for controlling the location and timing of gene activity. In addition, inducible versions of Cre recombinase, e.g., CreER, allow temporal control to the manipulation of gene activity, which becomes important when analyzing gene function at specific embryonic and adult stages (1,2). Promoters of the pancreas duodenal homeobox 1 (Pdx1) (3,4) and insulin (Ins1 and Ins2) (5–8) genes have been well characterized to allow the use of regulatory sequences for directing Cre expression to specific pancreatic cell populations. Commonly used transgenic mouse lines that employ rat Ins2 gene promoter sequences to drive Cre expression within the β-cell population include Ins2-Cre/RIP-Cre [Mouse Genome Informatics (MGI): Tg(Ins2-cre)^25Mgn and Tg(Ins2-cre)^1Herr] (9–11) and RIP-CreER [MGI: Tg(Ins2-cre/Esr1)^1Dam] (12). Pdx1 gene promoter sequences have proven useful for directing Cre expression throughout the early pancreatic epithelium (4,10,13,14) and to the endocrine cells of the pancreas (15). The Pdx1 gene is expressed early in pancreas development throughout the endoderm of the dorsal and ventral buds, but expression becomes restricted during development such that high levels of Pdx1 are maintained in the insulin-producing β-cells with lower levels in subpopulations of acinar cells (8,16). Examples of Pdx1-Cre transgenic lines include Pdx1-Cre^early [MGI: Tg(Pdx1-cre)^89.1Dam] (13), Pdx1-Cre^late [MGI: Tg(Ipf1-cre/Esr1)^1Dam/Mmcd] (10), Pdx1-Cre [MGI: Tg(Ipf1-cre)^1Tuv] (14), and Pdx1-CreER [MGI: Tg(Pdx1-cre/ERT)^1Mga] (15).To assess the specificity of recombination and perform lineage tracing analysis, reporter lines such as the ROSA26-stop-lacZ [MGI: Gt(ROSA)26Sor^tm1Sho], also known as R26R (17), or the ROSA26-stop-YFP [MGI: Gt(ROSA)26Sor^tm1(EYFP)Cos] (18) mice have been developed. Upon Cre-mediated recombination, these reporter lines activate expression of a β-galactosidase (β-gal) or a yellow fluorescent protein (YFP) reporter under the control of the ubiquitously active ROSA26 promoter, resulting in expression that is stably inherited by all cell progeny regardless of their differentiation fate.Here we show that most Cre lines currently being used to mediate pancreas or β-cell recombination also direct Cre expression to areas of the brain, and this may lead to altered gene expression in nutrient-sensing neurons that affects nutrient homeostasis.     

Pparg-P465L Mutation Worsens Hyperglycemia in Ins2-Akita Female Mice via Adipose-Specific Insulin Resistance and Storage Dysfunction

OBJECTIVE

The dominant-negative P467L mutation in peroxisome proliferator activated receptor-γ (PPARγ) was identified in insulin-resistant patients with hyperglycemia and lipodystrophy. In contrast, mice carrying the corresponding Pparg-P465L mutation have normal insulin sensitivity, with mild hyperinsulinemia. We hypothesized that murine Pparg-P465L mutation leads to covert insulin resistance, which is masked by hyperinsulinemia and increased pancreatic islet mass, to retain normal plasma glucose.

RESEARCH DESIGN AND METHODS

We introduced in Pparg^P465L/+ mice an Ins2-Akita mutation that causes improper protein folding and islet apoptosis to lower plasma insulin.

RESULTS

Unlike Ins2^Akita/+ littermates, male Pparg^P465L/+Ins2^Akita/+ mice have drastically reduced life span with enhanced type 1 diabetes. Hyperglycemia in Ins2^Akita/+ females is mild. However, Pparg^P465L/+Ins2^Akita/+ females have aggravated hyperglycemia, smaller islets, and reduced plasma insulin. In an insulin tolerance test, they showed smaller reduction in plasma glucose, indicating impaired insulin sensitivity. Although gluconeogenesis is enhanced in Pparg^P465L/+Ins2^Akita/+ mice compared with Ins2^Akita/+, exogenous insulin equally suppressed gluconeogenesis in hepatocytes, suggesting that Pparg^P465L/+Ins2^Akita/+ livers are insulin sensitive. Expression of genes regulating insulin sensitivity and glycogen and triglyceride contents suggest that skeletal muscles are equally insulin sensitive. In contrast, adipose tissue and isolated adipocytes from Pparg^P465L/+Ins2^Akita/+ mice have impaired glucose uptake in response to exogenous insulin. Pparg^P465L/+Ins2^Akita/+ mice have smaller fat depots composed of larger adipocytes, suggesting impaired lipid storage with subsequent hepatomegaly and hypertriglyceridemia.

CONCLUSIONS

PPARg-P465L mutation worsens hyperglycemia in Ins2^Akita/+ mice primarily because of adipose-specific insulin resistance and altered storage function. This underscores the important interplay between insulin and PPARγ in adipose tissues in diabetes.Diabetes is a major health care challenge in itself and significantly increases cardiovascular disease morbidity and mortality. As a multifactorial, chronic disease, diabetes emanates from the complex interaction of genetic and environmental influences. Among the many factors presumed or shown to contribute to its pathology, peroxisome proliferator activated receptor-γ (PPARγ) is an important candidate. PPARγ is a nuclear receptor and is necessary for adipocyte differentiation and triglyceride deposition (1). Activation of PPARγ has already provided therapeutic potential. One group of its synthetic ligands, the thiazolidinedione drugs, has found applications as antidiabetic agents (2).Various point mutations in PPARγ that affect adipose tissue distribution and insulin sensitivity have been identified in humans. For example, the PPARG-P12A polymorphism in humans is associated with reduced body weight and increased insulin sensitivity (3). Increased PPARγ activity in PPARG-P115Q mutation is associated with severe obesity and mild insulin resistance (4). Conversely, two dominant negative mutations resulting in decreased PPARγ activity, PPARG-P467L and PPARG-V290M, were reported in patients with severe insulin resistance (5). To date, different mouse models have demonstrated the role of PPARγ in varied metabolic processes. Lack of Pparg causes embryonic lethality in mice (6,7), and the Pparg-null embryos have no perceptible adipose tissue (6,8). Tissue-specific Pparg knockouts in liver (9) and skeletal muscle (10) exhibit insulin resistance. In contrast, absence of Pparg in β-cells does not affect glucose homeostasis, although it increases β-cell mass (11). Animals entirely lacking Pparg are nonviable, and tissue-specific knockouts offer a strategy for artificial manipulation that is only possible within experimental settings. Thus, an important step is to extend this work to study the role of PPARγ in a context applicable to human patients.The dominant-negative heterozygous PPARG-P467L mutation was originally identified in patients with severe insulin resistance, hyperglycemia, lipodystrophy, and hypertension (5). However, mice carrying the corresponding Pparg-P465L mutation (L/+ or L) exhibit normal plasma glucose and insulin sensitivity (12,13). These mice have mild hyperinsulinemia and increased pancreatic islet mass, especially on high-fat diet (12). In this study, we attempted to understand whether the observed insulin-resistance phenotype in human patients is indeed attributable to L/+ mutation or a mere association. We hypothesized that mice carrying the L/+ mutation have covert insulin resistance; however, the simultaneous occurrence of islet hyperplasia and hyperinsulinemia compensates for this insulin resistance to retain normal plasma glucose.Improper folding of insulin due to the C96Y Ins2-Akita mutation causes endoplasmic reticulum stress and β-cell apoptosis, leading to reduced plasma insulin (14). The mutation only affects β-cells, and the heterozygous Akita mice develop consistently elevated plasma glucose levels early in postnatal life. Consequently, Akita model is uniquely suited to test our prediction that, with reduced insulin production, the Pparg^P465L/+ mice would be unable to compensate for the peripheral insulin resistance. We show here that the Akita females with Pparg^P465L/+ mutation have increased severity of hyperglycemia and insulin resistance restricted to adipose tissue.     

The intercalated cells of the mouse kidney OMCDis are the target of the vasopressin V1a receptor axis for urinary acidification

Background

Vasopressin V1a receptor (V1aR) null mice have insufficient acid–base balance, but the target cell for V1aR signaling which results in the urinary acidification has not been identified.

Methods

By using a quantitative in situ hybridization technique and a double-staining technique with an anti-AQP3 antibody in mice, we investigated the axial distribution and acidosis-induced expression of V1aR mRNA along the nephron. We also investigated the acidosis-induced morphological change in the tubule cells from wild-type and V1aR-null (V1aR^?/?) mice.

Results

In the normal condition, V1aR mRNA was moderately expressed in the medullary thick ascending limb (MTAL) and highly expressed in the intercalated cell (IC) throughout the collecting duct (CD). However, no expression was observed in the proximal tubule, thin limbs of Henle’s loop, and the principal cell of the CD. Importantly, V1aR mRNA was upregulated significantly both in the TAL and the IC of the CD in the inner stripe of the outer medulla (MTAL_is and IC of OMCD_is, respectively) when mice were treated with NH₄Cl (0.28 mol/L) for 6 days. Acidosis-induced hypertrophy, which was completely attenuated in V1aR^?/? mice, was observed only in the IC of OMCD_is (P < 0.005). In addition, urinary excretion of ammonia (NH₃/NH₄ ⁺) was significantly decreased on day 3 (P < 0.05) and day 6 (P < 0.005) in the V1aR^?/? mice treated with NH₄Cl.

Conclusion

In conclusion, the IC of OMCD_is may be the target cell stimulated by the vasopressin V1aR axis and contribute to urinary acidification, at least during metabolic acidosis.     

Promoter Methylation of Specific Genes is Associated with the Phenotype and Progression of Colorectal Adenocarcinomas

Background

Promoter methylation of colorectal cancer-related genes were examined with respect to phenotype and tumor progression.

Materials and Methods

We assayed promoter methylation of 11 genes including established CpG island methylator phenotype (CIMP) markers (MLH1, MINT1, MINT2, MINT31, p16 ^INK4a , p14 ^ARF , and CACNA1G) and four genes (COX2, DAPK, MGMT, and APC) frequently methylated in colorectal cancer in 285 patients with sporadic colorectal cancer.

Results

CIMP+ tumors were more than two times more frequent among high-frequency microsatellite instability tumors (MSI-H) than in tumors without MSI (P ≤ .0001–.002). COX2 and DAPK methylation were significantly associated with CIMP+ and MSI. KRAS showed tendency toward more frequent codon 12-13 mutations identified in tumors with APC and p16 ^INK4a methylation than in those with unmethylation (P = .033 and .05, respectively). Additionally, tumors with synchronous adenoma were associated with p16 ^INK4a methylation (P = .004). The p16 ^INK4a methylation was significantly associated with poor overall and disease-free survival in 131 rectal cancer patients who underwent curative operation, according to multivariate analyses (relative risk [RR] = 0.317 and 0.349; P = .033 and .024, respectively). Specifically, in 175 stage II and III patients receiving adjuvant-based fluoropyrimidine chemotherapy, p16 ^INK4a methylation and MINT31 unmethylation showed a significant or tendency toward an association with recurrence and DFS (P = .007–.032).

Conclusions

The study suggests that specific CIMP markers, such as p16 ^INK4a and MINT31, should be further verified as potential epigenetic targets for the design of efficient chemotherapy regimens. We also identified a subset of colorectal cancer, possibly comprising APC methylation-KRAS mutation-p16 ^INK4a methylation.     

Receptor for Advanced Glycation End Products (RAGE) on iNKT Cells Mediates Lung Ischemia–Reperfusion Injury

Activation of invariant natural killer T (iNKT) cells and signaling through receptor for advanced glycation end products (RAGE) are known to independently mediate lung ischemia–reperfusion (IR) injury. This study tests the hypothesis that activation of RAGE specifically on iNKT cells via alveolar macrophage‐produced high mobility group box 1 (HMGB1) is critical for the initiation of lung IR injury. A murine in vivo hilar clamp model was utilized, which demonstrated that RAGE^?/? mice were significantly protected from IR injury. Treatment of WT mice with soluble RAGE (a decoy receptor), or anti‐HMGB1 antibody, attenuated lung IR injury and inflammation, whereas treatment with recombinant HMGB1 enhanced IR injury in WT mice but not RAGE^?/? mice. Importantly, lung dysfunction, cytokine production and neutrophil infiltration were significantly attenuated after IR in Jα18^?/? mice reconstituted with RAGE^?/? iNKT cells (versus WT iNKT cells). In vitro studies demonstrated that, after hypoxia‐reoxygenation, alveolar macrophage‐derived HMGB1 augmented IL‐17 production from iNKT cells in a RAGE‐dependent manner. These results suggest that HMGB1‐mediated RAGE activation on iNKT cells is critical for initiation of lung IR injury and that a crosstalk between macrophages and iNKT cells via the HMGB1/RAGE axis mediates IL‐17 production by iNKT cells causing neutrophil infiltration and lung IR injury.

     

Arterial wall hypertrophy is ameliorated by α2-adrenergic receptor antagonist or aliskiren in kidneys of angiotensinogen-knockout mice

Background

Arterial hypertrophy and interstitial fibrosis are important characteristics in kidneys of angiotensinogen-knockout (Atg ^?/?) mice. In these mice, which exhibit polyuria and hypotension, sympathetic nerve signaling is estimated to be compensatorily hyperactive. Furthermore, transforming growth factor (TGF)-β1 is overexpressed in mice kidneys. To determine whether sympathetic nerve signaling and TGF-β1 exacerbate arterial hypertrophy and interstitial fibrosis, intervention studies of such signaling are required.

Methods

We performed renal denervation and administered the α2-adrenergic receptor (AR) antagonist, atipamezole, to Atg ^?/? mice. A renin inhibitor, aliskiren, which was preliminarily confirmed to reduce TGF-β1 gene expression in kidneys of the mice, was additionally administered to assess the effect on the arterial hypertrophy and interstitial fibrosis.

Results

Norepinephrine content in kidneys of Atg ^?/? mice was three times higher than in kidneys of wild-type mice. Interventions by renal denervation and atipamezole resulted in amelioration of the histological findings. Overexpression of TGF-β1 gene in kidneys of Atg ^?/? mice was altered in a manner linked to the histological findings. Surprisingly, aliskiren reduced α2-AR gene expression, interstitial fibrosis, and arterial hypertrophy in kidneys of Atg ^?/? mice, which lack renin substrate.

Conclusions

Alpha2-AR signaling is one of the causes of persistent renal arterial hypertrophy in Atg ^?/? mice. Aliskiren also angiotensinogen-independently reduces the extent of renal arterial hypertrophy, partly thorough downregulation of α2-ARs. Although renal arterial hypertrophy in Atg ^?/? mice appears to be of multifactorial origin, TGF-β1 may play a key role in the persistence of such hypertrophy.

     

Age-Dependent Decline in ��-Cell Proliferation Restricts the Capacity of ��-Cell Regeneration in Mice

OBJECTIVE

The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of β-cell mass.

RESEARCH DESIGN AND METHODS

We analyzed the capacity of β-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration.

RESULTS

Young mice responded to high-fat diet by increasing β-cell mass and β-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in β-cell mass or β-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of β-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and β-cell proliferation was analyzed to assess the regeneration of β-cells. We observed a fourfold increase in β-cell proliferation in young mice after STZ administration, whereas no changes in β-cell proliferation were observed in older mice. The capacity to expand β-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of β-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16^Ink4aexpression in the β-cells. Young Bmi1^−/− mice that prematurely upregulate p16^Ink4afailed to expand β-cell mass in response to exendin-4, indicating that p16^Ink4alevels are a critical determinant of β-cell mass expansion.

CONCLUSIONS

β-Cell proliferation and the capacity of β-cells to regenerate declines with age and is regulated by the Bmi1/p16^Ink4apathway.Hyperglycemia in type 1 and 2 diabetes is, by definition, caused by insufficient insulin secretion to meet insulin demand. Defective insulin secretion in both forms of diabetes is caused in part by loss of β-cell mass (1–4). Diabetes can be reversed in type 1 and 2 diabetes by replacement of β-cell mass, as demonstrated by pancreas and islet transplantation (5,6). However, given the shortage of organ donors and the need for chronic immunosuppression, pancreas transplantation has limited applicability in the treatment of diabetes.Regeneration of β-cell mass is one promising approach to replace the deficit in β-cell mass in diabetic patients. Regeneration occurs in rodents after injury or genetic ablation of β-cells (7). Lineage tracing experiments show that new β-cells can arise from proliferation of preexisting β-cells (8). However, both the capacity of regeneration and the mechanism involved can differ significantly depending on the experimental model. An alternative source of β-cells has recently been proposed showing that facultative progenitors can be found in regenerating pancreatic ducts (9). Several studies have shown that the endocrine pancreas has endogenous renewal capacity in response to metabolic demands such as pregnancy and insulin resistance (10). Changes in insulin demand caused by physiological states such as insulin resistance have been shown to lead to adaptive changes in β-cell mass. The cell source and mechanism leading to the endogenous renewal is not clear, although proliferation of β-cells appears to play an important role (11). Elucidating mechanisms of regeneration and endogenous renewal in response to metabolic demands may provide novel insights into approaches to restore functional β-cell mass in diabetes.Most of the studies exploring the capacity of endogenous renewal have been carried out on rodents at relatively young ages, and several studies suggest that the capacity to expand or regenerate β-cell mass may decline with age. For example, a threefold increase of insulin content was measured in the residual pancreas after 90% pancreatectomy in 1-month-old rats; however, a comparable increase was not observed in rats that were 5 or 15 months old (12). Moreover, consistent with the adaptive increase in pancreatic insulin content in the 1-month-old but not older animals after a 90% pancreatectomy, blood glucose values in the 1-month-old rats declined 2 weeks after surgery, whereas no such decline was observed in 5- and 15-month-old rats (12). The rate of β-cell proliferation gradually declines with aging in rats to a steady state by 7 months of age (13). Furthermore, long-term bromodeoxyuridine labeling in 1-year-old mice also suggests that β-cell replication rates decline with age (14). The decline in β-cell proliferation with age correlates with increased expression of the cell cycle regulator p16^Ink4a in islet cells (15). p16^Ink4a inhibits the cyclin-dependent kinase 4 (CDK4)-cyclin D2 complex and can inhibit cell cycle progression and regeneration of islet cells. Transgenic mice that overexpressed p16^Ink4a showed reduced islet cell proliferation and a reduction in the regenerative capacity of islets after toxin-mediated destruction. However, the mechanisms that regulate the increase in p16^Ink4a with aging are not known.Establishing the basis of aging in affecting the capacity of adaptive changes in β-cell mass in adult humans versus young rodents has important clinical implications. If it is a species difference, then caution will need to be exercised extrapolating findings in rodents to the potential for β-cell regeneration in humans. For example, partial pancreatectomy in young mice is followed by extensive regeneration of β-cells through β-cell replication. In contrast, partial pancreatecomy in adult humans does not lead to β-cell regeneration (16). To date, it is not clear whether this different outcome is a species difference or a consequence of partial pancreatectomy at different ages. Also of interest, genetically obese mice have a several-fold increase in β-cell mass, whereas obese adult humans have only a much more modest 0.5-fold increase in β-cell mass (1). Again, it is not known whether this is a species difference or the consequence of a different response to obesity-induced insulin resistance during aging.Studies to date that have reported an increase in β-cell mass with glucagon-like peptide 1 (GLP-1)-1 based therapies were undertaken in young rodents (17–21). On the strength of those observations, it has been proposed that these therapies might serve to foster β-cell regeneration in humans with either type 1 or type 2 diabetes (22–25). However, it is plausible that GLP-1–induced expansion of β-cell mass may only be achievable in young subjects. Similarly, the rapid regeneration of β-cell mass in young mice after a single dose of the β-cell toxin streptozotocin (STZ) has been widely used as a model for β-cell regeneration in type 1 diabetes, but it is not yet known whether there is comparable recovery of β-cell mass in mice during the adult phase of β-cell turnover, a circumstance more clinically relevant to most humans.In the current studies, the capacity of endogenous renewal of β-cells in response to either a high-fat diet or the GLP-1 analog exendin-4 as well as the capacity to regenerate after toxin administration was examined in young and old mice. β-Cell mass and metabolic measurements were measured after high-fat diet or exendin-4 treatment. β-Cell proliferation was measured to probe the mechanism by which age could affect the capacity for β-cell renewal. Furthermore, the levels of p16^ink4a were linked to the capacity of β-cell proliferation. Because several studies have established a role for the polycomb group protein Bmi1 in the regulation of p16^Ink4a (26–28), we analyzed the levels of Bmi1. We further showed that loss of Bmi1 results in premature increase in the expression of p16^ink4a. Because aging can lead to many changes in the β-cell other than p16^Ink4a upregulation, we used Bmi1 knockout mice as a model to explore the endogenous capacity to renew in young mice in which p16^ink4a is prematurely upregulated. This result indicates that levels of p16^ink4a are the primary determinant of the capacity to expand β-cell mass. Our data suggest that the older mice, unlike younger mice, have a limited capacity to expand β-cell mass because of age-related accumulation of p16^Ink4a.     

Silencing of TLR4 Increases Tumor Progression and Lung Metastasis in a Murine Model of Breast Cancer

Background

Toll-like receptor 4 (TLR4) is a member of a family of pattern recognition receptors that are involved in the host defense against microbial infection. Little research has investigated the link between TLR4 and cancer. We thus addressed the effect of TLR4 in both the host immune system and cancer cells with regard to its effect on breast cancer progression and metastasis.

Methods

Adult female Balb/c mice aged 6–8 weeks were divided into three groups. In group 1, 15 each wild-type and TLR4^?/? mice were inoculated with 4T1 cells; in group 2, wild-type mice were inoculated with 4T1 cells (n = 15), 4T1 cells transduced with TLR4 lentivirus (n = 15) or with control lentivirus (n = 15); and in group 3, 15 TLR4^?/? mice were inoculated with 4T1 cells transduced with TLR4 lentivirus. Flank tumor volume was measured with calipers during weeks 2–5. Animals were then humanely killed and the number of macroscopic lung nodules counted.

Results

There was a significant increase in tumor volume in weeks 2, 3 and 4 after inoculation of TLR4^?/? mice with 4T1 cells compared with wild-type mice (p < 0.05). The number of metastatic lung nodules was significantly higher in TLR4^?/? mice (p < 0.05), and survival of tumor-bearing TLR4^?/? mice was substantially reduced compared with wild-type mice (p = 0.004). Knockdown of TLR4 from the 4T1 cells led to a relative reduction in lung metastasis, although it did not reach statistical significance.

Conclusions

TLR4 exerts both a defensive role at the host level and a negative role at the cancer cell level in this murine metastatic breast tumor model. Further evaluation of the role of TLR4 in breast cancer is warranted.

     

Endothelial nitric oxide synthase gene polymorphisms and the risk of osteonecrosis of the femoral head in systemic lupus erythematosus

Purpose

Nitric oxide (NO), a short-lived gaseous free radical, is a potent mediator of biological responses involved in the pathogenesis of autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Nitric oxide also serves as an important signal in physiological processes, including angiogenesis, thrombosis, and bone turnover, which are known to be related to the pathogenesis of osteonecrosis. We investigated whether NOS3 gene polymorphisms are associated with risk of osteonecrosis of the femoral head (ONFH).

Methods

Five polymorphisms in the NOS3 gene were genotyped using TaqMan assays in 306 controls, 150 SLE patients, and 50 SLE patients with ONFH (SLE_ONFH).

Results

We found that Asp258Asp and Glu298Asp (G894T) polymorphisms in the NOS3 gene were significantly associated with risk of ONFH. Additionally, we calculated haplotype frequencies of a linkage disequilibrium (LD) block in NOS3 (rs1799983???rs1800780) and tested for haplotype associations. The haplotypes G-A and T-A showed significant protective (P?=?1.6?×?10^-3; OR 0.39, 95 % confidence intervals (CI) 0.22–0.7) and increased risk (P?=?2.0 x 10^-5–6.0 x 10^-4; OR 3.17?3.73) effects for ONFH, respectively.

Conclusions

These results suggest that exonic NOS3 polymorphisms may increase the risk of ONFH in Korean SLE patients     

Systematic Review and Meta-Analysis Comparing the Surgical Outcomes of Invasive Intraductal Papillary Mucinous Neoplasms and Conventional Pancreatic Ductal Adenocarcinoma

Objective

The aim of this study was to summarize the current literature comparing the surgical outcomes of invasive intraductal papillary mucinous neoplasms (IPMN^INV) and conventional pancreatic ductal adenocarcinomas (PDAC) in order to determine the differences in disease characteristics and prognosis.

Methods

Systematic review of the literature yielded 12 comparative studies reporting the clinicopathological characteristics and overall survival (OS) of 1,450 patients with IPMN^INV with 19,304 patients with conventional PDAC.

Results

IPMN^INV had a significantly lower likelihood of tumors extending beyond the pancreas [27.6 vs. 94.3 %; T4 vs. T1: odds ratio (OR) 0.111, 95 % confidence intervals (CI) 0.057–0.214], nodal metastasis (45.4 vs. 62.9 %: OR 0.507, 95 % CI 0.347–0.741), positive margin (14.2 vs. 28.3 %; OR 0.438, 95 % CI 0.322–0.596), perineural invasion (49.2 vs. 76.5 %; OR 0.304, 95 % CI 0.106–0.877) and vascular invasion (25.2 vs. 45.7 % OR 0.417, 95 % CI 0.177–0.980) when compared with PDAC. The 5-year OS of IPMN^INV was significantly better than PDAC [31.4 vs. 12.4 %: hazard ratio (HR) 0.659, 95 % CI 0.574–0.756]. The tubular subtype had a poorer 5-year OS and demonstrated significantly more aggressive features such as nodal metastases, vascular invasion, and perineural invasion compared with the colloid subtype.

Conclusion

IPMN^INV were significantly more likely to present at an earlier stage and were less likely to demonstrate nodal involvement, perineural invasion and vascular invasion. When controlled for stage, IPMN^INV had an improved OS when compared with PDAC in the early stages.     

Effects of knockout of the receptor for advanced glycation end‐products on bone mineral density and synovitis in mice with intra‐articular fractures

Our group employed the mouse closed intra‐articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post‐traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end‐products (RAGE ^?/?) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post‐fracture. MicroCT (μCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post‐fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP‐3, ‐13) expression were evaluated by histologic analyses. In wild‐type mice, μCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE^?/? mice. Synovitis was significantly elevated in 84 mJ impact wild‐type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE^?/? mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture‐related increases in MMP‐3 and ?13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE ^?/? accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439–2449, 2018.