The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.     

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204^−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204^−/− mice compared to WT mice. Aortas and MRA of miR-204^−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP₃R1) as a target of miR-204. Aortas and MRA of miR-204^−/− mice had higher expression of IP₃R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204^−/− and WT mice was abolished with pharmacologic inhibition of IP₃R1. Furthermore, Ang II-induced aortic IP₃R1 was greater in miR-204^−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204^−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP₃R1, and boosted SR Ca²⁺ release in response to PE, while overexpression of miR-204 downregulated IP₃R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP₃R1 interaction rescued miR-204-induced downregulation of IP₃R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP₃R1-dependent regulation of SR calcium release.     

CD1d-restricted NKT cells modulate placental and uterine leukocyte populations during chlamydial infection in mice

Invariant CD1d-restricted natural killer T cells play an important immunoregulatory role and can influence a broad spectrum of immunological responses including against bacterial infections. They are present at the fetal–maternal interface and although it has been reported that experimental systemic iNKT cell activation can induce mouse abortion, their role during pregnancy remain poorly understood. In the present work, using a physiological Chlamydia muridarum infection model, we have shown that, in vaginally infected pregnant mice, C. muridarum is cleared similarly in C57BL/6 wild type (WT) and CD1d^−/− mice. We have also shown that infected- as well as uninfected-CD1d^−/− mice have the same litter size as WT counterparts. Thus, CD1d-restricted cells are required neither for the resolution of chlamydial infection of the lower-genital tract, nor for the maintenance of reproductive capacity. However, unexpected differences in T cell populations were observed in uninfected pregnant females, as CD1d^−/− placentas contained significantly higher percentages of CD4⁺ and CD8⁺ T cells than WT counterparts. However, infection triggered a significant decrease in the percentages of CD4⁺ T cells in CD1d^−/− mice. In infected WT pregnant mice, the numbers of uterine CD4⁺ and CD8⁺ T cells, monocytes and granulocytes were greatly increased, changes not observed in infected CD1d^−/− mice. An increase in the percentage of CD8⁺ T cells seems independent of CD1d-restricted cells as it occurred in both WT and CD1d^−/− mice. Thus, in the steady state, the lack of CD1d-restricted NKT cells affects leukocyte populations only in the placenta. In Chlamydia-infected pregnant mice, the immune response against Chlamydia is dampened in the uterus. Our results suggest that CD1d-restricted NKT cells play a role in the recruitment or homeostasis of leukocyte populations at the maternal–fetal interface in the presence or absence of Chlamydia infection.     

Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing

Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn^−/−) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and βigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn^−/−, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn^−/− fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn^−/− fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn^−/− cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn^−/− cells. Quantification of immunostaining for vinculin and integrinβ1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinβ1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.     

Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions,gut barrier dysfunction and liver damage in mice

BackgroundDisruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury.MethodsWild type (WT) and occludin deficient (Ocln^−/−) mice were fed 1–6% ethanol in Lieber–DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers.ResultsEthanol feeding significantly reduced body weight gain in Ocln^−/− mice. Ethanol increased inulin permeability in colon of both WT and Ocln^−/− mice, but the effect was 4-fold higher in Ocln^−/− mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln^−/− mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln^−/− mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln^−/− mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln^−/− mice.ConclusionThis study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

Cover Image,Volume 120, Number 11, November 2019

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).     

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σ_alb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σ_alb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H₂O₂. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H₂O₂ indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H₂O₂- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.     

Adipose monocyte chemotactic protein-1 deficiency reduces high-fat diet-enhanced mammary tumorigenesis in MMTV-PyMT mice

Monocyte chemotactic protein-1 (MCP-1) is an adipokine with demonstrated carcinogenic potential. However, there is a lack of evidence whether adipose-produced MCP-1 contributes to breast cancer. We tested the hypothesis that adipose-produced MCP-1 contributes to mammary tumorigenesis in this study. In a breast cancer model of mouse mammary tumor virus-polyomavirus middle T-antigen (MMTV-PyMT), mice with or without adipose MCP-1 knockout [designated as Mcp-1^−/− or wild-type (WT)] were fed the standard AIN93G diet (16% of energy from soybean oil) or a high-fat diet (HFD, 45% of energy from soybean oil). Adipose MCP-1 knockout reduced Mcp-1 expression in adipose tissue and concentrations of MCP-1 in plasma. Mcp-1^−/− mice fed the HFD had less body fat than their WT counterparts. Adipose MCP-1 knockout attenuated HFD-enhanced mammary tumorigenesis, evidenced by lower mammary tumor volume. Furthermore, Mcp-1^−/− mice, regardless of diet, had a longer tumor latency and less tumor weight than WT mice. When fed the HFD, Mcp-1^−/− mice, compared to WT mice, exhibited lower concentrations of insulin, leptin, resistin, vascular endothelial growth factor and hepatic growth factor in plasma. In summary, adipose MCP-1 deficiency attenuated HFD-enhanced MMTV-PyMT mammary tumorigenesis. This attenuation can be attributed to less body adiposity, improvement in insulin sensitivity and down-regulation in protumorigenic inflammation cytokines and angiogenic factors in Mcp-1^−/− mice. It concludes that adipose-produced MCP-1 contributes to mammary tumorigenesis in the MMTV-PyMT mouse model.     

Modulation of hepatic lipidome by rhodioloside in high-fat diet fed apolipoprotein E knockout mice

BackgroundRhodioloside is a glucoside of tyrosol isolated from Rhodiola rosea. However, its regulating effect on hepatic dyslipidemia of atherogenic mice has rarely been studied.PurposeThe specific aims of current study included to clarify lipidomic perturbation in liver tissues of apolipoprotein E deficient (apoE^−/−) mice fed with high-fat diet, and to examine the effects of rhodioloside against atherosclerosis and dyslipidemia.Study DesignThe comparisons of hepatic lipidome were executed between wide type (WT) mice fed with normal diet (NDC) and apoE^−/− mice fed with high-fat diet (Model), WT mice fed with high-fat diet (HFDC) versus the model mice, as well as the model mice versus rhodioloside-treated atherosclerotic mice.MethodsUltra high performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UPLC-MS/MS) was employed to provide an unbiased and simultaneous measurement of individual lipid species in liver tissues.ResultsMultivariate statistical analysis derived from LC-MS spectra revealed that high-fat diet and apoE deficiency caused a series of disturbances on glyerolipid metabolism, glycerophospholipid metabolism and sphingolipid metabolism. Rhodioloside administration showed atheroprotective effects on the apoE^−/− mice with regulating the levels of 1 phosphatidylcholine, 2 phosphatidylserines, 5 alkyldiacylglycerols and 3 alkenyldiacylglycerols back to normal. In particular, PC (4:0/15:0) was positively associated with high-density lipoprotein cholesterol in blood, both of which could be ameliorated by rhodioloside.ConclusionOur results identified the abnormal hepatic lipids in atherosclerosis progression that could efficiently improved by rhodioloside. These lipids contributed to biological understanding of atherogenic dyslipidemia in liver and could also served as sensitive indicators for drug target screening.     

SOCS2 modulates adipose tissue inflammation and expansion in mice

IntroductionObesity is usually triggered by a nutrient overload that favors adipocyte hypertrophy and increases the number of pro-inflammatory cells and mediators into adipose tissue. These mediators may be regulated by suppressors of cytokine signaling (SOCS), such as SOCS2, which is involved in the regulation of the inflammatory response of many diseases, but its role in obesity is not yet known. We aimed to investigate the role of SOCS2 in metabolic and inflammatory dysfunction induced by a high-refined carbohydrate-containing diet (HC).Material and methodsMale C57BL/6 wild type (WT) and SOCS2 deficient (SOCS2^−/−) mice were fed chow or an HC diet for 8 weeks.ResultsIn general, SOCS2 deficient mice, independent of the diet, showed higher adipose tissue mass compared with their WT counterparts that were associated with decreased lipogenesis rate in adipose tissue, lipolysis in adipocyte culture and energy expenditure. An anti-inflammatory profile was observed in adipose tissue of SOCS2^−/− by reduced secretion of cytokines, such as TNF and IL-6, and increased M2-like macrophages and regulatory T cells compared with WT mice. Also, SOCS2 deficiency reduced the differentiation/expansion of pro-inflammatory cells in the spleen but increased Th2 and Treg cells compared with their WT counterparts.ConclusionThe SOCS2 protein is an important modulator of obesity that regulates the metabolic pathways related to adipocyte size. Additionally, SOCS2 is an inflammatory regulator that appears to be essential for controlling the release of cytokines and the differentiation/recruitment of cells into adipose tissue during the development of obesity.     

Ecto-5′-nucleotidase (CD73) is a potential target of hepatocellular carcinoma

High expression of ecto-5′-nucleotidase (CD73) has been reported in a number of epithelium origin malignancies. Here, we hypothesize that CD73 promotes hepatocellular carcinoma (HCC) growth and metastasis and that the effect is mediated by epithelial growth factor receptor (EGFR). HCC cells with different malignancies and Tissue microarrays of the tumor and peritumoral liver tissues from 30 independent patients were used to examine CD73 and EGFR expression. Then, MTT and Ki67 detection, together with cell adhesion, invasion, and migration assays were used to evaluate the effects of CD73 on cell growth and metastasis. The expression of EGFR in HCC cells was also tested after suppressing or overexpressing CD73. Lastly, tumor tissues from nude mice, which had been injected subcutaneously with HCC cells, were transplanted subcutaneously into CD73^−/− and wild-type (WT) C57 mice. CD73 expression was higher in HCC cells with greater metastatic potentials and tumor tissues compared with low metastatic cells and peritumor tissues. CD73 and EGFR were coexpressed and positively correlated in tumor and peritumor liver tissues in HCC tissue microarrays. Up-regulationof CD73 by plasmid transfection or by pharmacological agents promoted EGFR expression in HCC cells, whereas suppression of CD73 inhibited these effects. The growth of transplanted tumor tissues was dramatically slower in CD73^−/− mice than in WT type mice in the in vivo experiments. CD73 promotes HCC growth and metastasis and upregulated the expression of EGFR in HCC. Thus, CD73 and EGFR are potential targets in the treatment of HCC.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

Endothelium-specific SIRT1 overexpression inhibits hyperglycemia-induced upregulation of vascular cell senescence

The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Mammalian sirtuin 1 (SIRT1) has been shown to decrease high-glucose-induced endothelial cell senescence in vitro and prevent hyperglycemia-induced vascular dysfunction. However, a role for SIRT1 in prevention of hyperglycemia-induced vascular cell senescence in vivo remains unclear. We used endothelium-specific SIRT1 transgenic (SIRT1-Tg) mice and wild-type (WT) mice to construct a 40-week streptozotocin (STZ)-induced diabetic mouse model. In this mode, 42.9% of wild-type (WT) mice and 38.5% of SIRT1-Tg mice were successfully established as diabetic. Forty weeks of hyperglycemia induced significant vascular cell senescence in aortas of mice, as indicated by upregulation of expression of senescence-associated markers including p53, p21 and plasminogen activator inhibitor-1 (PAI-1). However, SIRT1-Tg diabetic mice displayed dramatically decreased expression of p53, p21 and PAI-1 compared with diabetic WT mice. Moreover, manganese superoxide dismutase expression (MnSOD) was significantly downregulated in the aortas of diabetic WT mice, but was preserved in diabetic SIRT1-Tg mice. Furthermore, expression of the oxidative stress adaptor p66Shc was significantly decreased in aortas of SIRT1-Tg diabetic mice compared with WT diabetic mice. Overall, these findings suggest that SIRT1-mediated inhibition of hyperglycemia-induced vascular cell senescence is mediated at least partly through the reduction of oxidative stress.     

Characterization and potential roles of calretinin in rodent spermatozoa

Calretinin has been detected in various excitable cells but the presence and putative roles of such a calcium-binding protein has never been characterized in sperm.Epididymal spermatozoa were collected from C57Bl6 (wild-type, WT) or calretinin knockout (CR^−/−) mice and Wistar rats. A specific staining for calretinin was detected by immunofluorescence in the principal piece of the flagellum, both in WT mouse and rat spermatozoa. Western blots confirmed the expression of calretinin in rat and WT spermatozoa as well as its absence in CR^−/− mice.No significant difference was observed in the spontaneous acrosome reaction between WT and CR^−/− sperm. The addition of the calcium-ionophore A-23187, Thapsigargin or Progesterone to WT or CR^−/− incubated spermatozoa induced increases in the acrosome reaction but the stimulatory effects were identical in both genotypes. Motility measurements assessed by computer-assisted sperm analysis indicated that, under basal non-stimulatory conditions, CR^-/- sperm exhibited a lower curvilinear velocity and a smaller lateral head movement amplitude, although no difference was observed for the beat cross frequency. After incubation with 25 mM NH₄Cl, the curvilinear velocity, the amplitude of the lateral head movement and the hyperactivation were increased, while the beat cross frequency was decreased, in both genotypes.Evaluation of the in vivo fertility potential indicated that the CR^−/− litter sizes were clearly reduced compared to the WT litter sizes.Our study describes, for the first time, the expression of calretinin in sperm. These data extend the potential implication of calcium-binding proteins in the sperm calcium-signaling cascade and bring new insights into the understanding of sperm physiology.     

Transient Receptor Potential Canonical Type 3 Channels Control the Vascular Contractility of Mouse Mesenteric Arteries

Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca²⁺ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K⁺)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca²⁺]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K⁺- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca²⁺ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca²⁺ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries.