Double-null Mice Research Articles

Abstract Tu127: Very low-density lipoprotein receptor mediates triglyceride-rich lipoprotein induced oxidative stress and insulin resistance.

Background: Very-low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of triglyceride–rich lipoproteins. Although VLDLR is highly expressed in the heart, its role in VLDLR in oxidative stress and insulin resistance is unclear. Here, we used lean (WT), genetically obese leptin-deficient (ob/ob), and leptin–VLDLR double-null (ob/ob-VLDLR-/-) mice to determine the role of VLDLR in VLDL-induced oxidative stress and insulin resistance in the heart. Methods: Insulin resistance was investigated using glucose tolerance test and uptake of labeled deoxyglucose. Uptake VLDL was assessed in vivo and in isolated cardiomyocytes using double labeled ([3H]-TG, cholesteryl oleoyl ether ([14C])-VLDL. Oxidative stress was evaluated by the measurement of lipid peroxidation products (LPO) and hydrogen peroxides (HPO). NADPH-dependent superoxide production was measured in tissue homogenates and cardiomyocytes with the lucigenin chemiluminescent assay. Superoxide production in cardiomyocytes was investigated using fluorescent probe. Results: While insulin sensitivity and glucose uptake were reduced in the hearts of ob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition in the heart. This was accompanied by an upregulation of cardiac NADPH oxidase (Nox) expression (+100%), increased oxidative stress markers LPO and HPO (+300%) and Nox-dependent superoxide production (+300%). Silencing VLDLR in ob/ob mice had reduced VLDL uptake and prevented excess lipid deposition in the heart, in addition to a reduction of superoxide production and the normalization of insulin sensitivity and glucose uptake. In isolated cardiomyocytes, VLDLR deficiency had prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake. Conclusions: Our findings indicate that VLDLR deficiency prevents excess lipid accumulation and moderates oxidative stress and insulin resistance in the heart of obese mice. This effect is linked to the active role of VLDLR in VLDL uptake, which triggers a cascade of events leading to increased NOX activity, overproduction superoxide and insulin resistance.

Very low-density lipoprotein receptor mediates triglyceride-rich lipoprotein-induced oxidative stress and insulin resistance.

Obesity is associated with excess lipid deposition in nonadipose tissues, leading to increased oxidative stress and insulin resistance. Very low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of triglyceride-rich lipoproteins. Although VLDLR is highly expressed in the heart, its role in obesity-associated oxidative stress and insulin resistance is unclear. Here, we used lean (wild type), genetically obese leptin-deficient (ob/ob), and leptin-VLDLR double-null (ob/ob-VLDLR-/-) mice to determine the impact of VLDLR deficiency on obesity-induced oxidative stress and insulin resistance in the heart. Although insulin sensitivity and glucose uptake were reduced in the hearts of ob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition. This was accompanied by an upregulation of cardiac NADPH oxidase (Nox) expression and increased production of Nox-dependent superoxides. Silencing the VLDLR in ob/ob mice had reduced VLDL uptake and prevented excess lipid deposition in the heart, in addition to a reduction of superoxide overproduction and the normalization of insulin sensitivity and glucose uptake. In isolated cardiomyocytes, VLDLR deficiency had prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake. Our findings indicate that VLDLR deficiency prevents excess lipid accumulation and moderates oxidative stress and insulin resistance in the hearts of obese mice. This effect is linked to the active role of VLDLR in VLDL uptake, which triggers a cascade of events leading to increased Nox activity, superoxide overproduction, and insulin resistance.NEW & NOTEWORTHY Obesity is associated with excess lipid deposition in muscles, which is considered as a leading cause of metabolic dysfunction and oxidative stress. Cellular uptake of lipids is regulated by several membrane receptors, among which is the very low-density lipoprotein receptor (VLDLR). This article provides information on the role of VLDLR in cardiac muscle and how its expression regulates insulin resistance and oxidative stress in the obese mouse model.

Abstract 3076: Cd36 Restricts Lipid-associated Macrophages Accumulation In White Adipose Tissues During Atherogenesis

Visceral white adipose tissue (WAT) regulates systemic lipid metabolism and inflammation. Dysfunctional WAT drives chronic inflammation and facilitates atherosclerosis. Adipose tissue macrophages (ATMs) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATMs crosstalk with other cells, driving chronic inflammation. Here, we conducted single-cell RNA sequencing on major WAT ATM subpopulations using a diet-induced atherosclerotic mouse model ( Apoe -null). We also examined the role of CD36 using Apoe / Cd36 double-null mice. Our results indicate that ATMs are constantly under lipid stress. We identified lipid-associated macrophages (LAMs), which were previously described in obesity. Interestingly, LAMs increased 8.4-fold in Apoe / Cd36 double-null mice on an atherogenic diet, but not in Apoe -null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation, which indicate a delicate balance between lipid metabolism and inflammation, mediated through CD36.

Abstract A077: The EZH2 epigenetic factor orchestrates oncogenic properties in ATM-deficient pancreatic cancer

Abstract Despite decades of extensive basic and translational research improving our understanding of pancreatic ductal adenocarcinoma (PDAC), its complex biology remains a significant challenge and limits overcoming its dismal prognosis. Due to its resistance to conventional therapies and heterogeneous mutational landscape, molecular stratification and the development of targeted therapeutic options are essential for improving survival of PDAC patients. Previous work from our group investigated loss-of-function mouse models for the serine/threonine kinase ATM causing homologous recombination deficiency (HRD) in PDAC. Specifically, ATM loss caused certain oncogenic features in the arising tumors, including the acquisition of an invasive phenotype along with the emergence of a prominent desmoplastic reaction, but also sensitizes toward platinum-based therapies and PARP1 inhibition (Russell, Nat Commun; Perkhofer, Cancer Res; Gout, Gut). However, the mechanisms orchestrating these oncogenic features remain unclear. Here, we identified the histone-methyltransferase EZH2 as a direct ATM substrate to operate as an epithelial-mesenchymal gatekeeper. In detail, ATM-deficient tumors accumulated EZH2, suggesting that ATM status contributes to rewiring the epigenetic landscape of pancreatic tumors, subsequently supporting oncogenic features. To investigate whether and how EZH2 impacts ATM-deficient PDAC pathogenesis, we generated conditional double ATM and EZH2-knockout in the KrasG12D/+; Ptf1aCre/+ mice. Double-null mice showed extended mouse survival, reduced metastasis burden, and decreased undifferentiated malignant cell features. Interestingly, both in vivo and in vitro investigations revealed that ATM status significantly impacts EZH2 activity via a post-translational regulation axis. Specifically, the ATM kinase activity was critical to promote Culin1 E3-ubiquitin ligase recruitment and subsequent EZH2 ubiquitination and degradation. Finally, we demonstrated that a combinatorial therapy involving a potent EZH2 inhibitor promotes genomic instability and exacerbates the cytotoxic effects of PARP interference in ATM-deficient HRD PDAC. Overall, our study demonstrates that ATM deficiency significantly impacts EZH2 epigenetic functions and pinpoints the molecular stratification according to ATM status prior to pharmacological EZH2 interference in PDAC therapies as a valuable approach to target ATM-deficient HRD tumors. Citation Format: Elodie Roger, Anna Härle, Johann Gout, Lukas Perkhofer, Elisabeth Hessmann, Alexander Kleger. The EZH2 epigenetic factor orchestrates oncogenic properties in ATM-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A077.

The Functional Redundancy of Neddylation E2s and E3s in Modulating the Fitness of Regulatory T Cells.

Neddylation is necessary for activation of Cullin-RING ligases (CRLs), which degrade various immune regulatory proteins. Our recent study showed that while depletion of neddylation E2-E3 pair Ube2f-Sag in regulatory T (Treg) cells had no obvious phenotype, the same depletion of either Ube2m or Rbx1 caused inflammation disorders with different severity. Whether these E2s or E3s compensate each other in functional regulations of Treg cells is, however, previously unknown. In this report, we generated Foxp3Cre;Ube2mfl/fl;Ube2ffl/fl or Foxp3Cre;Rbx1fl/fl;Sagfl/fl double-null mice by simultaneous deletion of both neddylation E2s or E3s in Treg cells, respectively. Remarkably, Ube2m&Ube2f double-null mice developed much severe autoimmune phenotypes than did Ube2m-null mice, indicating that Ube2m markedly compensates Ube2f in Treg cells. The minor worsened autoimmune phenotypes seen at the very early stage in Rbx1&Sag double-null than Rbx1-null mice is likely due to already severe phenotypes of the later, indicating a minor compensation of Rbx1 for Sag. The RNA profiling-based analyses revealed that up- and down-regulations of few signaling pathways in Treg cells are associated with the severity of autoimmune phenotypes. Finally, severer inflammation phenotypes seen in mice with double E3-null than with double E2-null Treg cells indicate a neddylation-independent mechanism of 2 E3s, also known to serve as the RING component of CRLs in regulation of Treg cell fitness.

Lhb−/−Lhr−/− Double Mutant Mice Phenocopy Lhb−/− or Lhr−/− Single Mutants and Display Defects in Leydig Cells and Steroidogenesis

In the mouse, two distinct populations of Leydig cells arise during testis development. Fetal Leydig cells arise from a stem cell population and produce T required for masculinization. It is debated whether they persist in the adult testis. A second adult Leydig stem cell population gives rise to progenitor-immature-mature adult type Leydig cells that produce T in response to LH to maintain spermatogenesis. In testis of adult null male mice lacking either only LH (Lhb-/-) or LHR (Lhr-/-), mature Leydig cells are absent but fetal Leydig cells persist. Thus, it is not clear whether other ligands signal via LHRs in Lhb null mice or LH signals via other receptors in the absence of LHR in Lhr null mice. Moreover, it is not clear whether truncated LHR isoforms generated from the same Lhr gene promoter encode functionally relevant LH receptors. To determine the in vivo roles of LH-LHR signaling pathway in the Leydig cell lineage, we generated double null mutant mice lacking both LH Ligand and all forms of LHR. Phenotypic analysis indicated testis morpho-histological characteristics are identical among double null and single mutants which all showed poorly developed interstitium with a reduction in Leydig cell number and absence of late stage spermatids. Gene expression analyses confirmed that the majority of the T biosynthesis pathway enzyme-encoding mRNAs expressed in Leydig cells were all suppressed. Expression of thrombospondin-2, a fetal Leydig cell marker gene was upregulated in single and double null mutants indicating that fetal Leydig cells originate and develop independent of LH-LHR signaling pathway in vivo. Serum and intratesticular T levels were similarly suppressed in single and double mutants. Consequently, expression of AR-regulated genes in Sertoli and germ cells were similarly affected in single and double mutants without any evidence of any additive effect in the combined absence of both LH and LHR. Our studies unequivocally provide genetic evidence that in the mouse testis, fetal Leydig cells do not require LH-LHR signaling pathway and a one-to-one LH ligand-LHR signaling pathway exists in vivo to regulate adult Leydig cell lineage and spermatogenesis.

Plasma Membrane Localization of CD36 Requires Vimentin Phosphorylation; A Mechanism by Which Macrophage Vimentin Promotes Atherosclerosis.

Vimentin is a type III intermediate filament protein expressed in cells of mesenchymal origin. Vimentin has been thought to function mainly as a structural protein and roles of vimentin in other cellular processes have not been extensively studied. Our current study aims to reveal functions of vimentin in macrophage foam cell formation, the critical stage of atherosclerosis. We demonstrated that vimentin null (Vim–/–) mouse peritoneal macrophages take up less oxidized LDL (oxLDL) than vimentin wild type (Vim+/+) macrophages. Despite less uptake of oxLDL in Vim–/– macrophages, Vim+/+ and Vim–/– macrophages did not show difference in expression of CD36 known to mediate oxLDL uptake. However, CD36 localized in plasma membrane was 50% less in Vim–/– macrophages than in Vim+/+ macrophages. OxLDL/CD36 interaction induced protein kinase A (PKA)-mediated vimentin (Ser72) phosphorylation. Cd36–/– macrophages did not exhibit vimentin phosphorylation (Ser72) in response to oxLDL. Experiments using phospho-mimetic mutation of vimentin revealed that macrophages with aspartate-substituted vimentin (V72D) showed more oxLDL uptake and membrane CD36. LDL receptor null (Ldlr–/–) mice reconstituted with Vim–/– bone marrow fed a western diet for 15 weeks showed 43% less atherosclerotic lesion formation than Ldlr–/– mice with Vim+/+ bone marrow. In addition, Apoe–/–Vim–/– (double null) mice fed a western diet for 15 weeks also showed 57% less atherosclerotic lesion formation than Apoe–/– and Vim+/+mice. We concluded that oxLDL via CD36 induces PKA-mediated phosphorylation of vimentin (Ser72) and phosphorylated vimentin (Ser72) directs CD36 trafficking to plasma membrane in macrophages. This study reveals a function of vimentin in CD36 trafficking and macrophage foam cell formation and may guide to establish a new strategy for the treatment of atherosclerosis.

Gaseotransmitters Modulate Inspiratory Drive from the Hypoglossal Nucleus

Gaseotransmitters, carbon monoxide (CO) and hydrogen sulfide (H2S), play an important role in maintaining proper function of cardio‐respiratory systems. Previous work has demonstrated that Heme Oxygenase‐2 knock out mice (HO‐2‐/‐) develop a sleep apnea phenotype characterized predominantly by obstructive apneas, which could be normalized by either treating HO‐2‐/‐ with L‐propargylglycine (L‐PAG) an inhibitor of cystathionine gamma‐lysase (CSE) and H2S synthesizing enzyme or in HO‐2+ CSE double null mice. Obstructive apneas are often caused by a loss in upper airway tone, we sought to examine how H2S and CO signaling impacts inspiratory activity from the preBötC and the hypoglossal nucleus (XIIn). We hypothesized that HO‐2 and CSE are involved in the regulation of inspiratory motor output from XIIn. Immunohistochemical analysis revealed many ChAT+ cells of XIIn are positive for HO‐2 and CSE. Biochemical analysis showed elevated H2S abundance in XIIn of HO‐2‐/‐mice. Electrophysiological recordings demonstrated transmission failure from preBötC to XIIn in response to NaHS, a H2S donor or Cr (III) Mesoporphyrin IX chloride (ChrMP459), an inhibitor of HO in brain slices from wild type mice. Furthermore, ChrMP459 reduced inspiratory drive currents received by XIIn neurons and suppressed excitability in XIIn neurons. Brain slices from HO‐2‐/‐mice showed: (1) a larger rate of transmission failure between preBötC and XIIn motor pools; and (2) smaller drive currents from individual XIIn neurons. Brain slices from HO‐2+ CSE double null mice displayed reduced transmission failures from preBötC to XIIn. Furthermore, L‐PAG mitigated transmission failures and increased the magnitude of inspiratory drive currents in XIIn neurons in HO‐2‐/‐mice. These findings demonstrate that HO‐2 and CSE regulate inspiratory drive from XIIn and suggest potential role for gaseotransmitters in regulating upper airway tone.

Olfactory Receptor78 Participates in Carotid Body‐Dependent Sympathetic Activation and Hypertension in Murine Models of Obstructive Sleep Apnea

Olfactory receptor (Olfr) 78 participates in carotid body (CB) activation by acute hypoxia. We examined the role of Olfr78 in CB‐dependent sympathetic activation and hypertension in: 1) mice treated with chronic intermittent hypoxia (CIH) patterned after blood O2 profiles during obstructive sleep apnea (OSA) and 2) heme oxygenase‐2 (HO‐2) null mice, which exhibit OSA. CIH treated wild type (WT) mice showed: enhanced CB sensory nerve response to hypoxia and sensory long‐term facilitation (sLTF), hypertension, activation of the sympathetic nervous system as evidenced by spectral analysis of inter‐beat interval as well as elevated plasma nor‐epinephrine (NE) levels. All these responses were absent in Olfr78 null mice. HO‐2 null mice showed spontaneous apneas with an apnea index (AI) of 58±1.2 apneas/hour compared to AI of 8±0.8 apneas/hour in WT mice. CBs of HO‐2 null mice showed enhanced CB response to hypoxia and sLTF as well as elevated plasma NE levels and hypertension. The magnitude of hypertension correlated to AI in HO‐2 null mice (P<0.01). In striking contrast, HO‐2/Olfr78 double null mice showed stable breathing with an AI of 12±1.3 apneas/hour. HO‐2/Olfr78 double null mice showed absence of augmented CB response to hypoxia, sLTF, elevated plasma NE levels and hypertension. These results demonstrate that Olfr78 participates in CB activation and ensuing sympathetic nerve excitation and hypertension in two mouse models of OSA.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

TLT-1, a Potential Regulator of Inflammatory Pathogenesis in Obesity and Non-Alcoholic Fatty Liver Disease (NAFLD)

Background:Obesity, a nationwide health issue, has related medical costs ranging between $147-210 billion per year in United States and has been associated with a 3.5-fold increased risk of developing NAFLD. In obesity, platelets work in a pleotropic manner with vascular and immune cells to amplify the chronic inflammatory process. Interestingly, studies have demonstrated that platelet numbers and reactivity are increased in obese individuals. The emerging role of activated platelets during obesity induced inflammation introduces the novel concept of platelet targeted therapeutic interventions.Kopec et al, further supports the idea that the mechanism underlying the progression of obesity lies in a platelet mediated pro-inflammatory state, illustrating that there is extravascular fibrin(ogen) deposition, macrophages and inflammatory cytokines within white adipose tissue and liver of mice on western diet. Kopec et al uses a fibrinogen mutant mouse (Fiby390-396 ) which lacks the binding motif for Mac-1 and inhibits the ligand interaction with leukocytes, diminishing inflammation, reducing macrophage counts, reducing weight, protects mice from NAFLD and glucose dysmetabolism. Taken together, all evidence points towards a platelet/fibrinogen/leukocyte pathophysiological mechanism underlying the development of obesity.TREM-Like Transcript-1 (TLT-1) is a platelet specific receptor found in the a-granules of platelets and released to the surface upon platelet activation. TLT-1 is a type 1 receptor that, like the integrin a2bb3, binds fibrinogen and facilitates platelet aggregation . However, although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory/nonimmune mediated setting, TLT-1's main association is with regulating inflammatory-derived bleeding. This is demonstrated by increased hemorrhage after inflammatory treatments such as lipopolysaccharide LPS in the treml1 -/- mice as compared to controls.Considering the emerging evidence in support of a platelet-fibrinogen receptor ligand interaction as a key mechanism underlying the development of obesity and that TLT-1, a platelet specific receptor binds fibrinogen and mediates leukocyte trafficking, our laboratory set out to determine whether TLT-1 could be implicated as the main culprit underlying this mechanism. When placed on a western diet, treml1 -/- mice are more prone to weight gain, based on these finding we hypothesize that: The TLT-1/Fibrinogen molecular interaction regulates metabolic inflammation in obesityAims:Evaluate the effects of western diet on obesity and NAFLD in the treml1 -/- mouse modelMethods:TLT-1 (treml1 -/-) - apolipoprotein E (apoe -/-) double null (AT-DKO;n=11) mice and control apoe +/-/treml1 +/- littermate controls (AT-Hets;n=20) were fed western diet for 20 weeks. Plasma samples were collected for adipokine, glucose, insulin, liver enzyme and lipid profiling. Mouse were perfused, liver and adipose tissue were collected for histological analysis.Results:Overall AT-DKO mice gained more weight compared to AT-Hets (12.94±1.90 vs 8.51±1.70 grams p=0.02). Plasma analysis demonstrates that the AT-DKO have higher levels of TNF-a (0.54±0.60 vs 0.118±0.17 pg/ml p=0.03), and IL-10 (2.50±1.40 vs 1.50±2.10 pg/ml p=0.004) compared to littermate controls. Histological analysis of livers illustrates increased lipid vacuoles and inflammatory foci in the AT-DKO mice as compared to controls, while preliminary data is not significant for these differences, liver damage in the AT-DKO was significantly greater as demonstrated by increased AST levels (166.21±91.00 vs 102±68.10 U/L p=0.02). Moreover, the AT-DKO mice had higher levels of ALT, direct bilirubin, cholesterol, pai-1 , triglycerides and lower IL-6 and Adiponectin (Table 1 data not significant). These findings suggest that in the absence of TLT-1 these mice are more prone to liver disfunction , hyperlipidemia and inflammatory alterations.Conclusions:Mutant AT-DKO mice are more prone to obesity and NAFLD compared to littermate controls, suggesting that TLT-1, a platelet gene, plays a surprising role in metabolism. Further investigation could adjudicate TLT-1 administration as a potential therapeutic intervention for prevention and amelioration of Obesity and related pathologies. The current state of this project will be reported here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functional cooperation between two otoconial proteins Oc90 and Nox3.

Otoconia-related vertigo and balance deficits are common in humans, but the molecular etiology is unknown at present. In order to study mechanisms of otoconia formation and maintenance, we have investigated whether otoconin-90 (Oc90), the predominant otoconial constituent protein, and the NADPH oxidase Nox3, an essential regulatory protein for otoconia formation, are functionally interlinked. We performed balance behavioral, electrophysiological, morphological and molecular cellular analyses. Double heterozygous mutant mice for Oc90 and Nox3 show severe imbalance, albeit less profound than double null mutants. In contrast, single heterozygous mutant mice have normal balance. Double heterozygous mice have otoconia defects and double null mice have no otoconia. In addition, some hair bundles in the latter mice go through accelerated degeneration. In vitro calcification analysis in cells stably expressing these proteins singly and doubly shows much more intense calcification in the double transfectants. Oc90 and Nox3 augment each other's function, which is not only critical for otoconia formation but also for hair bundle maintenance.

Role of olfactory receptor78 in carotid body-dependent sympathetic activation and hypertension in murine models of chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread breathing disorder. CIH-treated rodents exhibit activation of the sympathetic nervous system and hypertension. Heightened carotid body (CB) activity has been implicated in CIH-induced hypertension. CB expresses high abundance of olfactory receptor (Olfr) 78, a G-protein coupled receptor. Olfr 78 null mice exhibit impaired CB sensory nerve response to acute hypoxia. Present study examined whether Olfr78 participates in CB-dependent activation of the sympathetic nervous system and hypertension in CIH-treated mice and in hemeoxygenase (HO)-2 null mice experiencing CIH as a consequence of naturally occurring OSA. CIH-treated wild-type (WT) mice showed hypertension, biomarkers of sympathetic nerve activation, and enhanced CB sensory nerve response to hypoxia and sensory long-term facilitation (sLTF), and these responses were absent in CIH-treated Olfr78 null mice. HO-2 null mice showed higher apnea index (AI) (58 ± 1.2 apneas/h) than WT mice (AI = 8 ± 0.8 apneas/h) and exhibited elevated blood pressure (BP), elevated plasma norepinephrine (NE) levels, and heightened CB sensory nerve response to hypoxia and sLTF. The magnitude of hypertension correlated with AI in HO-2 null mice. In contrast, HO-2/Olfr78 double null mice showed absence of elevated BP and plasma NE levels and augmented CB response to hypoxia and sLTF. These results demonstrate that Olfr78 participates in sympathetic nerve activation and hypertension and heightened CB activity in two murine models of CIH.NEW & NOTEWORTHY Carotid body (CB) sensory nerve activation is essential for sympathetic nerve excitation and hypertension in rodents treated with chronic intermittent hypoxia (CIH) simulating blood O2 profiles during obstructive sleep apnea (OSA). Here, we report that CIH-treated mice and hemeoxygenase (HO)-2-deficient mice, which show OSA phenotype, exhibit sympathetic excitation, hypertension, and CB activation. These effects are absent in Olfr78 null and Olfr78/HO-2 double null mice.

O16: TARGETING THE LOX-1 SCAVENGER RECEPTOR ATTENUATES ATHEROSCLEROSIS AND NEOINTIMAL HYPERPLASIA IN APO-E NULL TRANSGENIC MICE

Abstract Introduction The clinical consequences of atherosclerosis and post-procedure neointimal hyperplasia (NIH) represent a significant proportion of the vascular surgeon's workload. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a class E scavenger receptor expressed in endothelial cells implicated in atherosclerosis and NIH. This study aims to demonstrate the therapeutic potential of LOX-1 targeting using a transgenic mouse model and LOX-1-specific non-antibody synthetic protein scaffolds called affimers. Method Apolipoprotein-E (APO-E) null and APO-E/LOX-1 double-null transgenic mice were fed a high cholesterol western diet for 12 weeks. One group of APO-E null mice was treated subcutaneously with LOX-1 affimer. All mice underwent unilateral common femoral artery wire injury at week 8. Mice were culled and tissues harvested for evaluation of atherosclerotic burden and femoral artery NIH. Studies were carried out with Home Office approval. Result Compared with APO-E null controls, carotid and femoral bifurcation atherosclerosis (p<0.05) and total aortic plaque coverage (p<0.01) were significantly reduced in APO-E/LOX-1 double-null mice. Reductions were observed in APO-E null mice treated with LOX-1 affimer, however these were non-significant. Femoral artery NIH was reduced by 16% in APO-E/LOX-1 double null and 11% in LOX-1 affimer treated APO-E null mice compared with untreated APO-E controls, however these reductions were non-significant. Conclusion This study demonstrates the therapeutic potential of LOX-1 targeting in large vessel atherosclerotic disease and NIH. Knockout of the LOX-1 transgene produced promising results, while results following LOX-1 affimer therapy were less impressive. Improvements in affimer pharmacokinetics and drug delivery optimisation are avenues for future studies. Take-home message The LOX-1 scavenger receptor is a potential novel therapeutic target in large-vessel atherosclerosis and neointimal hyperplasia.

Gaseous transmitter regulation of hypoxia-evoked catecholamine secretion from murine adrenal chromaffin cells.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H2S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H2S.NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H2S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

E2F1 regulates testicular descent and controls spermatogenesis by influencing WNT4 signaling.

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

Mice lacking DUSP6/8 have enhanced ERK1/2 activity and resistance to diet-induced obesity

Extracellular signal-regulated kinase 1 and 2 (ERK1/2) have been implicated as important regulators of metabolic homeostasis. Here we generated a new mouse model with genetic deletion of two ERK1/2 phosphatases, dual specificity phosphatase (DUSP) 6 and 8, to further define the role of ERK1/2 in obesity development. Dusp6/8 double-null mice demonstrated elevated ERK1/2 phosphorylation in multiple tissues, without any change of phosphorylation of p38 and c-Jun N-terminal kinases (JNKs). Elevated ERK1/2 activity in Dusp6/8 double-null mice was associated with larger hearts and other organs, consistent with greater rate of cell proliferation in these mice. However, ERK1/2 activation was not sufficient to protect the mouse hearts from pathological hypertrophy and interstitial fibrosis following angiotensin II and phenylephrine stimulation. Interestingly, mice lacking DUSP6/8 were resistant to high-fat diet-induced obesity. Serum triglyceride, lipid content in the liver and visceral adipose tissues was also dramatically reduced in Dusp6/8 double-null mice. Furthermore, Dusp6/8 double-null mice had improved glucose tolerance. Mechanistically, we found out that elevated ERK1/2 activity increased the expression levels of genes involved in lipid metabolism and glucose homeostasis. Together, our data suggest that ERK1/2 play an essential role for the management of metabolic homeostasis.

Haploinsufficiency of tau decreases survival of the mouse model of Niemann-Pick disease type C1 but does not alter tau phosphorylation.

Niemann-Pick C1 (NPC1) mouse models show neurofibrillary tangles as do human patients. A previous study in NPC1/tau double-null mutant mice showed that tau knockout nulls and heterozygotes unexpectedly had decreased survival when compared with NPC1 single mutants (Pacheco et al., Hum Molec Genetics 18:956-965, 2009). This was done in a null model of NPC1 (Npc1-/-). We have extended these results to a hypomorphic model (Npc1nmf164) and additionally studied tau phosphorylation, which has not been previously done in a tau heterozygote. As before, NPC1/tau double-mutant mice had shortened survival when compared with the NPC1 single mutant. Tau dosage was not affected by the Npc1 mutation. The increased phosphorylation of tau-ser396 previously noted in NPC1 mouse models was also present, but unaffected by the tau knockout, indicating that changes in tau phosphorylation are not the cause of decreased survival in NPC1/tau double mutants. Thus, the reason for shortened survival of NPC1 mouse models with concomitant tau haploinsufficiency is uncertain.

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia.

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (Pi ) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn-/- ) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn-/- mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn-/- mice were fed a high-Pi diet, serum Pi concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-Pi diet, potentially indicating a role for elevated inhibitory pyrophosphate (PPi ) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum Pi , immunohistochemistry for OPN on Hyp mice fed a high-Pi diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum Pi may be attributable to the well-known upregulation of mineralization-inhibiting OPN by Pi , and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic Pi -regulating factors. © 2020 American Society for Bone and Mineral Research.

Vitamin A Metabolism During Refractory Telogen

Abstract Objectives Hair follicles cycle through periods of growth (anagen), regression (catagen) and rest (telogen). Telogen is further divided into refractory and competent telogen based on the expression of bone morphogenetic protein 4 (BMP4). Previously, the expression of a complete set of proteins involved in retinoic acid (RA) synthesis and signaling localized to the hair follicle and changed throughout the hair cycle. In addition, excess dietary vitamin A arrested the hair cycle in telogen; while retinol dehydrogenases short-chain dehydrogenase/reductase family 16C members 5 and 6 (Sdr16c5−/−/Sdr16c6−/−) double null mice had an accelerated the hair cycle. The purpose of this study was to further define these changes in the hair cycle. Methods The localization of RA synthesis proteins SDR16C5, retinol dehydrogenase 10 (RDH10), retinal dehydrogenase 2 (ALDH1A2), cellular RA binding protein 2 (CRABP2), RA degradation enzyme cytochrome p450 26B1 (CYP26B1), and BMP4 was examined in telogen hair follicles in female C57BL/6 J mice by immunohistochemistry. Immunohistochemistry with an antibody against BMP4 was also used to mark refractory telogen in the previous dietary vitamin A study. Results All proteins localized to BMP4 positive refractory telogen hair follicles. SDR16C5 and ALDH1A2 were also seen in BMP4 negative competent telogen hair follicles, but at a lower level. RDH10 was expressed in both BMP4 negative and positive hair follicles at similar levels. BMP4 expression was also used to distinguish refractory from competent telogen in C57BL/6 J mice fed different levels of vitamin A. Both low and excess dietary vitamin A resulted a greater percentage of hair follicles in refractory telogen in different studies. Conclusions In conclusion, RA synthesis and signaling may be stronger in refractory telogen and contribute to the inhibition of the hair cycle. Funding Sources NIH/NIAMS, Internal funding.