Receptor Double Knockout Mice Research Articles

Human ApoC3 overexpression aggravates hyperlipidemia but mitigates diet-induced coronary atherosclerotic disease in SR-BI and LDL receptor double knockout mice

Human ApoC3 overexpression aggravates hyperlipidemia but mitigates diet-induced coronary atherosclerotic disease in SR-BI and LDL receptor double knockout mice

Loss of Farnesoid X receptor (FXR) accelerates dysregulated glucose and renal injury in db/db mice.

End-stage renal disease is primarily caused by diabetic kidney disease (DKD). The Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has anti-inflammatory, lipid-lowering and hypoglycemic properties. It also inhibits renal fibrosis. Although its physiological role is not fully understood, it also plays a role in the control of diabetic nephropathy (DN). In the present study, we examined male FXR & leptin receptor double knockout mice, in which weight, blood glucose, body fat, and other indicators were monitored. After 6 months of rearing, blood and urine samples were collected and biochemical parameters were measured. Fibrosis was assessed by Masson's stain, while the assessment of the resuscitation case's condition was performed using succinate dehydrogenase (SDHA) stain immunohistochemistry, which measures aerobic respiration. Expression of molecules such as connective tissue growth factor (CTGF), SMAD family members 3 (Smad3) and 7 (Smad7), and small heterodimer partner were detected by RT-PCR and Western blotting as part of the application. FXR knockout decreased body weight and body fat in db/db mice, but increased blood glucose, urine output, and renal fibrosis. Primary mesangial cells (P-MCs) from FXR+/+ mice stimulated with transforming growth factor β1 (TGFβ1) showed significantly higher levels of related fibrosis factors, TGFβ1 and Smad3 mRNA and protein, and significantly reduced levels of Smad7. These effects were reversed by the action of FXR agonist chenodeoxycholic acid (CDCA). P-MCs from FXR-/- mice stimulated with TGFβ1 resulted in an increase in the expression and protein levels of collagen I and TGFβ1, and the addition of CDCA had no significant effect on TGFβ1 stimulation. However, compared with FXR+/+db/db mice, the rate of oxygen consumption, the rate of carbon dioxide production, and the rate of energy conversion were increased in FXR-/-db/db mice, whereas the SDHA succinate dehydrogenase, a marker enzyme for aerobic respiration, was significantly decreased. These results provide evidence that FXR plays a critical role in the regulation of mesangial cells in DN. The likely mechanism is that aberrant FXR expression activates TGFβ1, which induces extracellular matrix accumulation through the classical Smad signaling pathway, leading to mitochondrial dysfunction.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification.

To evaluate the role and therapeutic value of homocysteine (hcy)-inducible endoplasmic reticulum stress (ERS) protein with ubiquitin like domain 1 (Herpud1) in hcy-induced calcific aortic valve disease (CAVD). The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. In vivo, we use the low-density lipoprotein receptor (LDLR) and Herpud1 double knockout (LDLR-/-/Herpud1-/-) mice and used high methionine diet (HMD) to assess of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs). In vitro, the role of Herpud1 in the Hcy-related osteogenic differentiation of AVICs was investigated by manipulating of Herpud1 expression. Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs). Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression. Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo. Herpud1 silencing activated cell autophagy, which subsequently inhibited hcy-induced osteogenic differentiation of AVICs. ERS inhibitor 4-phenyl butyric acid (4-PBA) significantly attenuated aortic valve calcification in HMD-fed low-density lipoprotein receptor-/- (LDLR-/-) mice by suppressing ERS and subsequent Herpud1 biosynthesis. These findings identify a previously unknown mechanism of Herpud1 upregulation in Hcy-related CAVD, suggesting that Herpud1 silencing or inhibition is a viable therapeutic strategy for arresting CAVD progression. • Herpud1 is upregulated in the leaflets of Hcy-treated mice and patients with CAVD. • In mice, global knockout of Herpud1 alleviates aortic valve calcification and Herpud1 silencing activates cell autophagy, inhibiting osteogenic differentiation of AVICs induced by Hcy. • 4-PBA suppressed Herpud1 expression to alleviate AVIC calcification in Hcy treated AVICs and to mitigate aortic valve calcification in mice.

1313-P: Short-Chain Fatty Acids Decrease Food Intake through Free Fatty Acid Receptors in the Brain

The influence of gut bacteria on host energy homeostasis is increasingly recognized, but mechanistic links are lacking. The gut microbiota digests and ferments nutrients, which result in short-chain fatty acids (SCFAs) . The body senses these nutrients in large part through free fatty acid receptors 2 (FFA2) and 3 (FFA3) . Accumulating evidence indicates that the gut microbiota/SCFAs interact with the central nervous system (CNS) to regulate brain metabolic function. Consistently, we found that a single acute intracerebroventricular (ICV) injection of acetate (ACE) , propionate (PRO) , or butyrate (BUT) dose-dependently inhibited food intake in an FFA2/3-dependent manner. The reduced food intake induced by ACE, PRO, or BUT was attributed to decreased meal size or increased intermeal interval. These regulatory effects on meal patterns were blunted in FFA2 and FFA3 double-receptor knockout mice, suggesting a mediating role of brain FFA2/3. Interestingly, RNAscope analysis showed that FFA3 but not FFA2 highly expressed in the cerebellar granule neurons. Using brain slice patch-clamp, we consistently showed that PRO or FFA3-selective agonists inhibited these granule neurons. Notably, the hyperpolarization effects of PRO on cerebellar granule neurons were blocked by pertussis toxin (PTX) , a G-protein coupled receptor subunit Gi/o blocker, further supporting an FFA3-Gi/o-mediated inhibition on granule neurons. These results suggest a model that SCFAs inhibit food intake through FFA3 expressed by the cerebellar granule neurons. Key words: SCFA; Brain; FFA3 Disclosure P. Luo: None. P. Xu: None. H. Ye: None. B.T. Layden: None. Y. He: None. V.C. Torres Irizarry: None.

Chronic ethanol consumption plus an atherogenic diet cause metabolic steatohepatitis with advanced liver fibrosis in apolipoprotein E/low-density lipoprotein receptor double-knockout mice.

Nonalcoholic steatohepatitis is the inflammatory subtype of nonalcoholic fatty liver disease with a high risk of progression to liver fibrosis. We investigated metabolic steatohepatitis with advanced liver fibrosis in apolipoprotein E/low-density lipoprotein receptor double-knockout (AL) mice fed a co-diet of ethanol with a low-carbohydrate-high-protein-high-fat atherogenic diet (AD) for 16weeks. We also examined the underlying mechanisms, especially hepatic sympathetic activation, involved in the effects. We maintained 12-week-old male AL mice on AD and a standard chow diet (SCD) with or without ethanol treatment for 16weeks. Age-matched male C57BL/6J mice on SCD without ethanol treatment served as controls. We conducted blood biochemical, histopathological, and fluorescence immunohistochemical, and reverse transcriptase polymerase chain reaction studies. AL mice showed significant hyperlipidemia. AD induced increased body weight, hepatic steatosis, and hepatic damage; ethanol and the AD co-diet resulted in hepatic sympathetic activation accompanied by hepatic steatosis, lobular inflammation, bridging fibrosis, and hepatic damage. Hepatic Kupffer cells (KCs) and hepatic stellate cells (HSCs), which showed sympathetic activation, produced 4.4- to 9.4-fold more inflammatory factors (KC and KC-derived tumor necrosis factor-α, and chemokine [C-C motif] ligand 2) and 2.0- to 32.0-fold more fibrosis factors (HSC and HSC-derived transforming growth factor β1 and collagen 1a1); all p<0.05 vs. controls. We created a model of metabolic steatohepatitis with advanced liver fibrosis from coexisting hyperlipidemia and hepatic sympathetic activation in AL mice on a co-diet of ethanol and AD. KCs and HSCs became the cellular targets of hepatic sympathetic activation, which could play a role in the initiation and progression of metabolic steatohepatitis with advanced liver fibrosis.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

Ablation of Galectin-12 Inhibits Atherosclerosis through Enhancement of M2 Macrophage Polarization

The formation of foam cells, which are macrophages that have engulfed oxidized low-density lipoprotein (OxLDL), constitutes the first stage in the development of atherosclerosis. Previously, we found that knocking down galectin-12, a negative regulator of lipolysis, leads to reduced secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that plays an important role in atherosclerosis. This prompted us to study the role of galectin-12 in atherosclerosis. With that aim, we examined foam cell formation in Gal12‒/‒ murine macrophages exposed to OxLDL and acetylated LDL (AcLDL). Then, we generated an LDL receptor and galectin-12 double knockout (DKO) mice and studied the effect of galectin-12 on macrophage function and atherosclerosis. Lastly, we evaluated the role of galectin-12 in human THP-1 macrophages using a doxycycline-inducible conditional knockdown system. Galectin-12 knockout significantly inhibited foam cell formation in murine macrophages through the downregulation of cluster of differentiation 36 (CD36), and the upregulation of ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1), and scavenger receptor class B type 1 (SRB1). Consistent with this, galectin-12 knockdown inhibited foam cell formation in human macrophages. In addition, the ablation of galectin-12 promoted M2 macrophage polarization in human and murine macrophages as evidenced by the upregulation of the M2 marker genes, CD206 and CD163, and downregulation of the M1 cytokines, tumor necrosis factor α (TNF- α), interleukin-6 (IL-6), and MCP-1. Moreover, the ablation of galectin-12 decreased atherosclerosis formation in DKO mice. Based on these results, we propose galectin-12 as a potential therapeutic target for atherosclerosis.

Alcohol consumption combined with dietary low-carbohydrate/high-protein intake increased the left ventricular systolic dysfunction risk and lethal ventricular arrhythmia susceptibility in apolipoprotein E/low-density lipoprotein receptor double-knockout mice.

Alcohol abuse is positively associated with cardiovascular disease. Dietary low-carbohydrate/high-protein (LCHP) intake confers a greater mortality risk. Here, the impact of ethanol consumption in combination with dietary LCHP intake on left ventricular (LV) systolic function and lethal ventricular arrhythmia susceptibility were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (AL) mice. The underlying mechanisms, cardiac sympathovagal balance, beta-adrenergic receptor (ADRB) levels, and gap junction channel protein connexin 43 (Cx43) expression, were examined. Male AL mice fed an LCHP diet with or without ethanol were bred for 16 weeks. Age-matched male AL and wild-type mice received standard chow diet and served as controls. The following were used to assess LV systolic function, lethal ventricular arrhythmia susceptibility, cardiac sympathovagal balance, Cx43 expression, and ADRB levels: The results demonstrated that ethanol consumption in combination with dietary LCHP intake worsened LCHP-induced LV systolic dysfunction in AL mice and enhanced their susceptibility in the ventricular arrhythmia-evoked test. There were concomitant increases in LV weight, LF/HF ratio shown by HRV, TH, ADRB1, ADRB2, and Cx43 expressions by LV fluorescence immunohistochemistry, and LV Cx43 messenger ribonucleic acid expression by PCR. In AL mice, alcohol consumption combined with dietary LCHP intake may thus promote a shift in cardiac sympathovagal balance toward sympathetic predominance, the increases in beta-adrenergic receptors (ADRB1 and ADRB2), and then affect the gap junction channel protein Cx43, which in turn could contribute to increased risks of LV systolic dysfunction and susceptibility to lethal ventricular arrhythmia.

Electrophysiological responses to sugars and amino acids in the nucleus of the solitary tract of type 1 taste receptor double-knockout mice.

Strong evidence supports a major role for heterodimers of the type 1 taste receptor (T1R) family in the taste transduction of sugars (T1R2+T1R3) and amino acids (T1R1+T1R3), but there are also neural and behavioral data supporting T1R-independent mechanisms. Most neural evidence for alternate mechanisms comes from whole nerve recordings in mice with deletion of a single T1R family member, limiting conclusions about the functional significance and T1R independence of the remaining responses. To clarify these issues, we recorded single-unit taste responses from the nucleus of the solitary tract in T1R double-knockout (double-KO) mice lacking functional T1R1+T1R3 [KO1+3] or T1R2+T1R3 [KO2+3] receptors and their wild-type background strains [WT; C57BL/6J (B6), 129X1/SvJ (S129)]. In both double-KO strains, responses to sugars and a moderate concentration of an monosodium glutamate + amiloride + inosine 5'-monophosphate cocktail (0.1 M, i.e., umami) were profoundly depressed, whereas a panel of 0.6 M amino acids were mostly unaffected. Strikingly, in contrast to WT mice, no double-KO neurons responded selectively to sugars and umami, precluding segregation of this group of stimuli from those representing other taste qualities in a multidimensional scaling analysis. Nevertheless, residual sugar responses, mainly elicited by monosaccharides, persisted as small "sideband" responses in double-KOs. Thus other receptors may convey limited information about sugars to the central nervous system, but T1Rs appear critical for coding the distinct perceptual features of sugar and umami stimuli. The persistence of amino acid responses supports previous proposals of alternate receptors, but because these stimuli affected multiple neuron types, further investigations are necessary.NEW & NOTEWORTHY The type 1 taste receptor (T1R) family is crucial for transducing sugars and amino acids, but there is evidence for T1R-independent mechanisms. In this study, single-unit recordings from the nucleus of the solitary tract in T1R double-knockout mice lacking T1R1+T1R3 or T1R2+T1R3 receptors revealed greatly reduced umami synergism and sugar responses. Nevertheless, residual sugar responses persisted, mainly elicited by monosaccharides and evident as "sidebands" in neurons activated more vigorously by other qualities.

Periovulatory insulin signaling is essential for ovulation, granulosa cell differentiation, and female fertility.

Recent studies have demonstrated an essential role for insulin signaling in folliculogenesis as conditional ablation of Igf1r in primary follicles elicits defective follicle-stimulating hormone responsiveness blocking development at the preantral stage. Thus the potential role of insulin action in the periovulatory window and in the corpus luteum is unknown. To examine this, we generated conditional Insr,Igf1r, and double receptor knockout mice driven by Pgr-Cre. These models escape the preantral follicle block and in response to superovulatory gonadotropins exhibit normal distribution of ovarian follicles and corpora lutea. However, single ablation of Igf1r leads to subfertility and mice lacking both receptors are infertile. Double knockout mice have impaired oocyte development and ovulation. While some oocytes are released and fertilized, subsequent embryo development is retarded, and the embryos potentially fail to thrive due to lack of luteal support. In support of this, we found reduced expression of key enzymes in the steroid synthesis pathway and reduced serum progesterone. In addition to metabolic and steroidogenic pathways, RNA-sequencing analysis revealed transcription factor-3 as an important transcription factor downstream of insulin signaling. Collectively, these results highlight the importance of growth factors of the insulin family during two distinct windows of follicular development, ovulation, and luteinization.

D-amino acid oxidase contributes IgA nephropathy through regulation of IgA production in intestinal mucosa

Abstract Gut mucosa protects our body against threats of bacteria using antimicrobial proteins such as defensins, cathelicidins or lysozyme. D-amino acid oxidase (DAO) is one of the antimicrobial protein that is released from goblet cells into lumen and catabolize D-amino acids, which is essential for almost all bacteria, into antibacterial agent; H2O2. IgA is major immunoglobulin in gut. Maturation of B cells into IgA-secreting plasma cells are lead through two pathways; T cell dependent or independent mechanism. A number of reports indicated the accumulation of systemic IgA in the animals lacking antimicrobial proteins. However, the mechanism how loss of innate immunity affects acquired immunity is yet to be clarified. We found that mouse lacking DAO activity showed accumulation of systemic IgA. Analysis of intestinal microbiota in DAO null mice revealed increased amount of specific bacteria that stimulated IgA production. Crossbreeding experiment between DAO null mice and T cell receptor double knockout (Tcrb−/−, Tcrd−/−) mice showed that the hyper production of IgA is caused by T cell dependent B cell activation. IgA nephropathy (IgAN) occasionally causes chronic kidney impairment. Recent genome wide association analysis indicated the linkage between IgAN and genes associated with mucosal immunity. We found that high IgA (HIGA) mouse, an animal model of IgAN, lacked DAO activity, and that high plasma IgA in HIGA mice is dependent on intestinal microbiota. These results indicated that loss of DAO activity involves in pathogenesis of IgAN through disturbance of mucosal immunity.

Gpihbp1 deficiency accelerates atherosclerosis and plaque instability in diabetic Ldlr-/- mice

Background and aimsGlycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) plays a crucial role in triglyceride hydrolysis, and GPIHBP1 deficiency leads to severe hypertriglyceridemia (HTG). Gpihbp1 knockout (GKO) mice develop mild lesions in the aortic root at the age of 11 months. Herein, we investigated the effect of Gpihbp1 deficiency on atherosclerosis (AS) under diabetic conditions. MethodsFor experiment 1, diabetes was induced in GKO and wild-type (WT) mice by injection of streptozotocin at 3 months of age and lasted for 4 months. For experiment 2, diabetes was induced in Gpihbp1/low-density lipoprotein receptor (Ldlr) double-knockout (GLDKO) mice, Ldlr knockout (LKO) mice were used as controls. The experiment was continued for 3 or 5 months. Plasma glucose and lipid levels were measured, and atherosclerotic lesions were analyzed at 3 and 5 months during the experiment. ResultsNo atherosclerotic lesions were detected in the aorta in GKO mice after 4 months of diabetes. Compared with LKO mice, GLDKO mice manifested enhanced aortic atherosclerotic lesions, decreased plaque stability, and increased oxidative stress and inflammation in plaques at 3 and 5 months after diabetes. Atherosclerotic lesions in the coronary artery and dilated remodeling in the aortic root were also found in GLDKO diabetic mice. ConclusionsGpihbp1 deficiency accelerates the development of AS in the aorta, and the instability of plaques in LKO mice and diabetes promotes these pathologic processes with coronary AS. These findings were probably associated with HTG caused by Gpihbp1 deficiency and with increased oxidative stress and inflammation in the atherosclerotic lesions.

Alcohol Consumption in Combination with an Atherogenic Diet Increased Indices of Atherosclerosis in Apolipoprotein E/Low-Density Lipoprotein Receptor Double-Knockout Mice.

Alcohol abuse and adherence to atherogenic diet (AD; a low-carbohydrate-high-protein diet) have been positively associated with cardiovascular disease. In addition, it has been demonstrated clinically that dietary intake is increased on days when alcohol is consumed. Here, the additive effects of ethanol (EtOH) and AD on atherosclerosis, a major underlying cause of cardiovascular disease, were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (KO) mice. The mechanisms, especially aortic oxidative stress damage, were highlighted. Twelve-week-old male KO mice on AD with or without EtOH treatment were bred for 4months. Age-matched male C57BL/6J mice on a standard chow diet without EtOH treatment served as controls. Analyses were conducted using ultrasound biomicroscopy, histopathological and fluorescence immunohistochemical examinations, Western blots, and polymerase chain reaction. KO mice on AD with EtOH treatment showed increases in aortic maximum intima media thickness, hypoechoic plaque formation, and mean Oil-Red-O content. These results were associated with enhanced ratio of aortic 8-hydroxy-2'-deoxyguanosine (8-OHdG)-immunopositive area to the metallothionein (MT) immunopositive area and suppression of AD-induced up-regulated aortic Mt1, Mt2, and upstream stimulatory factor 1 mRNA expressions. Moreover, 8-OHdG was expressed in the nuclei of CD31- and alpha smooth muscle actin-immunopositive cells, and the up-regulated mRNA expressions of aortic nitric oxide synthase 3 and platelet-derived growth factors were only observed in the KO mice on AD with EtOH treatment. Alcohol abuse and adherence to AD may promote the shift of aortic oxidative stress and antioxidative stress balance toward oxidative stress predominance and reduced antioxidative stress, which may be partly due to the decrease in MT at the cell biological level and down-regulation of Mt at the gene level, which in turn could play a role in the up-regulation of endothelial dysfunction-related and vascular smooth muscle cell proliferation-related gene expression and the progression of atherosclerosis in mice with hyperlipidemia.

Low testosterone in ApoE/LDL receptor double-knockout mice is associated with rarefied testicular capillaries together with fewer and smaller Leydig cells

The testis as a site for atherosclerotic changes has so far attracted little attention. We used the apolipoprotein E (ApoE)/low density lipoprotein (LDL) receptor deficient mouse model (KO) for atherosclerosis (20, 40, 60 and 87-week-old) to investigate whether Leydig cells or the capillary network are responsible for reduced serum testosterone levels previously observed in extreme ages of this model. In KO mice, overall testosterone levels were reduced whereas the adrenal gland-specific corticosterone was increased excluding a general defect of steroid hormone production. In addition to micro-CT investigations for bigger vessels, stereology revealed a reduction of capillary length, volume and surface area suggesting capillary rarefaction as a factor for diminished testosterone. Stereological analyses of interstitial cells demonstrated significantly reduced Leydig cell numbers and size. These structural changes in the testis occurred on an inflammatory background revealed by qPCR. Reduced litter size of the KO mice suggests hypo- or infertility as a consequence of the testicular defects. Our data suggest reduced testosterone levels in this atherosclerosis model might be explained by both, rarefication of the capillary network and reduced Leydig cell number and size. Thus, this study calls for specific treatment of male infertility induced by microvascular damage through hypercholesterolemia and atherosclerosis.

Surprising outcomes in cannabinoid CB1/CB2 receptor double knockout mice in two models of ischemia

AimsWe tested the hypothesis that CB1/CB2 receptor double knockout would produce significant increases in infarct size and volume and significant worsening in clinical score, using two mouse models, one of permanent ischemia and one of ischemia/reperfusion. Main methodsFocal cerebral infarcts were created using either photo induced permanent injury or transient middle cerebral artery occlusion. Infarct volume and motor function were evaluated in cannabinoid receptor 1/cannabinoid receptor 2 double knockout mice. Key findingsThe results surprisingly revealed that CB1/CB2 double knockout mice showed improved outcomes, with the most improvements in the mouse model of permanent ischemia. SignificanceAlthough the number of individuals suffering from stroke in the United States and worldwide will continue to grow, therapeutic intervention for treatment following stroke remains frustratingly limited. Both the cannabinoid 1 receptor (CB1R) and the cannabinoid 2 receptor (CB2R) have been studied in relationship to stroke. Deletion of the CB2R has been shown to worsen outcome, while selective CB2R agonists have been demonstrated to be neuroprotective following stroke. Although initial studies of CB1R knockout mice demonstrated increased injury following stroke, indicating that activation of the CB1R was neuroprotective, later studies of selective antagonists of the CB1R also demonstrated a protective effect. Surprisingly the double knockout animals had improved outcome. Since the phenotype of the double knockout is not dramatically changed, significant changes in the contribution of other homeostatic pathways in compensation for the loss of these two important receptors may explain these apparently contradictory results.

Characterization of spontaneous hydrocephalus development in the young atherosclerosis-prone mice.

Little has been reported on whether abnormal lipid metabolism affects hydrocephalus, although congenital malformations and infectious diseases are major causal factors for hydrocephalus development. In a study on the pathogenesis of atherogenesis in mice, we unexpectedly discovered that hydrocephalus occurred in partial apolipoptotein E (apoE) and low-density lipoprotein receptor (LDLR) double-knockout (apoE/LDLR) mice fed either chow or a high-fat and high-cholesterol diet between the ages of 4 and 12 weeks. In the 12-week-old high-fat and high-cholesterol group, the incidence rate was as high as 15%. Transcription levels of transforming growth factor-β1 (TGF-β1), Smad3, Smad4, and Smad7 in the cortex of the hydrocephalic cerebrum were significant downregulated in 4-week-old mice, but were increased in the 8 and 12-week-old groups compared with that of age-matched nonhydrocephalic mice. The mRNA level of tissue inhibitor of metalloproteinases 1 was significantly increased, whereas matrix metalloproteinase-9 was lower in hydrocephalic mice of all ages. The translation level of TGF-β1 increased in the hydrocephalic brains of 8 and 12-week-old mice. This study provides primary evidence for the connection between lipid metabolic disorder and hydrocephalus development. This may suggest that both hyperglyceridemia and hypercholesterolemia are harmful factors in hydrocephalus development because of adverse effects on TGF-β1/Smad signaling in the brain.

178 - Acetaldehyde Dehydrogenase 2(ALDH2) in Atherosclerosis: Beyond Detoxification of Reactive Aldehydes

Previous studies have linked human polymorphisms of ALDH2 to cardiovascular diseases but the underlying mechanisms remain elusive. Here we investigated the role of ALDH2 in atherosclerosis using a murine model of low density lipoprotein receptor (LDLR) and ALDH2 double knockout mice. Surprisingly, we found that double KO mice had significantly decreased areas of atherosclerotic plaque and less macrophages in mouse aorta comparing to LDLR KO mice. Bone marrow transplant experiments also supported an important role of macrophages in this phenotype. Moreover, in vitro experiments using bone marrow derived macrophages (BMDM) showed that macrophages from ALDH2 KO had decreased uptake of ox-LDL through downregulation of phagocytic genes in LDLR-/-background. CO-IP, IF experiments, and RNA-seq in macrophages demonstrated that LDLR regulated ALDH2 entering into nucleus and regulated genes related to autophagy, AMPK activation, and mitochondrial functions. Our findings shed light on novel roles of ALDH2 in the pathogenesis of atherosclerosis through modulating macrophages functions by interactions of LDLR and AMPK - beyond the conventional roles of detoxification of reactive aldehydes.

OP.1B.01] IMPLICATION OF THE BENEFICIAL EFFECT BY MAS RECEPTOR DEFICIENCY IN VASCULAR DEMENTIA

Objective: Regulation of renin-angiotensin-system (RAS) is expected to prevent the onset and progression of dementia. Angiotensin (Ang) converting enzyme (ACE) 2/Ang-(1–7)/Mas receptor axis has been highlighted as counteracting partner against the classical RAS, ACE/Ang II/Ang II type 1 (AT1) receptor axis. We recently reported that ACE2 deficiency caused deterioration of cognitive function. Mas receptor expression is abundant in hippocampus; however, the roles of Mas receptor in cognitive function are still an enigma. Our previous report indicates the role of Ang-(1–7) as a ligand for Ang II type 2 (AT2) receptor, which is known to improve cognitive function. Thus, we examined the possible roles of Mas receptor employing mouse vascular dementia model focusing on the interaction with AT2 receptor.Design and method: Male 10-week-old C57BL6 mice (wild-type, WT), Mas1 receptor knockout mice (MasKO) and AT2/Mas1 receptor double knockout mice (DKO) were subjected to bilateral carotid artery stenosis (BCAS) surgery. Six weeks after the surgery, we evaluated their cognitive functions with the Morris water maze test. Cerebral blood flow (CBF) was analyzed with laser speckle flowmetry. We also compared the effects of intraperitoneally administrated Ang-(1–7) in WT, MasKO, AT2KO and DKO on cognitive function. Results: There was no significant difference in spatial leaning memory between WT and MasKO in the Morris water maze test after BCAS surgery; however, DKO showed more impaired cognitive function than MasKO (Figure 1). CBF did not show any significant differences in each BCAS group. Administration of Ang-(1–7) did not change significantly these parameters in all BCAS group; however, Ang-(1–7) treatment tended to improve cognitive function even in MasKO mice (Figure 2). Conclusions: Our results suggested the possible protective effect of Mas receptor deficiency on vascular dementia. Administration of Ang-(1–7) could not improve the cognitive function under the deletion of AT2 receptor. Therefore, the interaction of Ang-(1–7) with AT2 receptor could contribute to maintain the cognitive function in MasKO.

Abstract P233: Mas Receptor Deficiency Does Not Impair Cognitive Funcion of Vascular Dementia Model in the Presence of Angiotensin II Type 2 Receptor

Objective: Angiotensin (Ang) converting enzyme (ACE) 2/ Ang-(1-7)/Mas receptor axis has been considered as protective arm in the renin-angiotensin system and Ang-(1-7) is thought to interact with Ang II type 2 (AT 2 ) receptor according. Mas receptor is expressed highly in hippocampus and blood vessels in brain, but its actual function is still unclear. Thus, we examined the possible roles of Mas receptor in relation to the vascular cognitive impairment focusing on the interaction with AT 2 receptor. Design and Methods: Male 10-week-old C57BL6 mice (wild-type, WT), Mas1 receptor knockout mice (MasKO) and AT 2 /Mas1 receptor double knockout mice (DKO) were subjected to bilateral carotid artery stenosis (BCAS) surgery. After six weeks from the treatment, we evaluated their cognitive function with Y-maze test and the Morris water maze test. Results: The cerebral blood flow (CBF) in each BCAS group was significantly reduced compared to its sham-operated counterparts (WT; 31.5±0.6 vs 28.0±0.8, MasKO; 31.7±0.8 vs 27.7±0.8, DKO; 33.0±0.5 vs 29.1±0.7). The alternation behavior (%) was significantly reduced in WT mice with BCAS compared to sham mice (69.6±3.5 vs 57.9±2.1), but there was no significant difference in MasKO and DKO mice (MasKO; 64.1±2.5 vs 63.1±2.5, DKO; 67.6±2.1 vs 61.1±4.0) in Y maze test. In the Morris water maze test, the mean arrival time at platform at day 5 (sec) was significantly higher in WT-BCAS mice than WT-sham mice (Sham; 20.9±4.6 vs BCAS; 47.3±6.5). In contrast to the results in WT, there was no significant difference in MasKO mice (Sham; 32.8±8.5 vs BCAS; 34.5±7.3). DKO-sham mice showed significantly lower spatial learning ability compared with WT-sham mice (DKO; 77.4±11.9 vs WT; 20.9±4.6). The total cell count in dentate gyrus area was significantly lower in WT-BCAS compared to WT-sham (sham; 255.7±7.0 vs BCAS; 209.4±5.4), but there was no significant change in MasKO mice(sham; 256.5±2.5 vs BCAS; 233.2±16.4). We could not see significant difference in the number of DCX-positive cells and the expressions of proinflammatory cytokines such as IL-6, TNF-α and MCP-1in all mouse groups. Conclusion: Mas receptor deficiency seems to be beneficial in vascular dementia on condition that AT 2 receptor exists.