Global Deletion Research Articles

359-OR: 12/15-Lipoxygenase in Islets and Macrophages Independently Promotes Autoimmune Diabetes in NOD Mice

The pathogenesis of type 1 diabetes (T1D) involves the interaction of the immune system with β cells. 12/15-lipoxygenase (12/15-LOX), an enzyme involved in arachidonic acid metabolism, is expressed in both β cells and macrophages. Global deletion of the gene encoding 12/15-LOX (Alox15) prevents diabetes in the NOD mouse model of T1D via reduction in production of proinflammatory mediators in islets and macrophages. However, the importance of 12/15-LOX in islets vs. macrophages remains unclear. To address this issue, we generated both inducible islet-cell-specific (NOD-Alox15∆islet) and myeloid-lineage-specific (Alox15∆myel) Alox15 knockout mice on the NOD background. Control female mice (Alox15loxp/loxp, Pdx1PB-CreERTM+, and LysM-Cre+) exhibited diabetes incidence greater than 75%, whereas both Alox15∆islet and Alox15∆myel showed a dramatic decrease in the incidence of diabetes (<10%, P<0.05). During the pre-diabetic phase (8 weeks of age), both control and Alox15∆islet mice exhibited similar β-cell mass (0.70±0.08 g vs. 0.72±0.06 g, P=NS) and insulitis severity (score 1.89±0.17 vs. 1.74±0.19, P=NS), whereas Alox15∆myel mice had increased β-cell mass (1.56±0.02c g, P<0.05) and insulitis (score 1.18±0.08, P<0.05). We next examined the nature of immune cells present in the spleen and pancreatic lymph node (pLN). Alox15∆islet mice showed increased Tregs (CD4+FoxP3; P<0.05) with no changes in Th1 (CD4+IFNγ+) and Th17 (CD4+IL17+) cells in both the spleen and pLN compared to control mice (P=NS). Additionally, Alox15∆islet female mice exhibited decreased IL-1β production by dendritic cells. By contrast, Alox15∆myel mice showed only decreased pLN proinflammatory macrophages (F480+IL1β+) and dendritic cells (CD11c+IL1β+) compared to controls (P<0.05). Together, these data suggest that 12/15-LOX functions independently in both the islet and macrophage through different mechanisms, promoting the destruction of β cells during the pathogenesis of autoimmune T1D. Disclosure A. Pineros Alvarez: None. A. Kulkarni: None. K. Orr: None. L. Glenn: None. C.A. Reissaus: None. M. Hernandez-Perez: None. M. Gannon: None. M.J. McDuffie: Stock/Shareholder; Self; Adenosine Therapeutics, LLC. J. Nadler: None. R. Mirmira: None. M.A. Morris: Stock/Shareholder; Spouse/Partner; Sanofi US. S.A. Tersey: None. Funding National Institutes of Health (R01DK105588 to R.M., J.N.)

Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.

The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.

Global deletion of Optineurin results in altered type I IFN signaling and abnormal bone remodeling in a model of Paget's disease.

Genome-wide association studies (GWAS) have identified Optineurin (OPTN) as genetically linked to Paget's disease of the bone (PDB), a chronic debilitating bone remodeling disorder characterized by localized areas of increased bone resorption and abnormal bone remodeling. However, only ~10% of mouse models with a mutation in Optn develop PDB, thus hindering the mechanistic understanding of the OPTN-PDB axis. Here, we reveal that 100% of aged Optn global knockout (Optn-/-) mice recapitulate the key clinical features observed in PDB patients, including polyostotic osteolytic lesions, mixed-phase lesions, and increased serum levels of alkaline phosphatase (ALP). Differentiation of primary osteoclasts ex vivo revealed that the absence of Optn resulted in an increased osteoclastogenesis. Mechanistically, Optn-deficient osteoclasts displayed a significantly decreased type I interferon (IFN) signature, resulting from both defective production of IFNβ and impaired signaling via the IFNα/βR, which acts as a negative feedback loop for osteoclastogenesis and survival. These data highlight the dual roles of OPTN in the type I IFN response to restrain osteoclast activation and bone resorption, offering a novel therapeutic target for PDB. Therefore, our study describes a novel and essential mouse model for PDB and define a key role for OPTN in osteoclast differentiation.

Function of Protein Kinase CK2 in Innate Immune Cells in Neuroinflammation

Abstract Immune dysregulation is critical to the pathogenesis of autoimmune diseases, including multiple sclerosis (MS). Studies in MS patients and an animal model of MS, experimental autoimmune encephalomyelitis (EAE), have highlighted the importance of both innate and adaptive immune responses in MS/EAE pathology. Antigen-presenting cells of the innate immune system, especially dendritic cells (DCs), not only activate naive CD4+ T cells by antigen presentation, but also control T cell differentiation by expressing co-stimulatory molecules and cytokines. Our current studies demonstrate that Tamoxifen-induced global deletion of CK2a in CK2αfl/flERT2Cre mice results in delayed onset and significantly slower progression of EAE, which is not observed in CK2α T cell-specific deleted CK2αfl/fldLckCre mice, which exhibit lower EAE clinical scores during the peak and resolution phases of disease. EAE mice with global CK2α deletion express reduced pathogenic Th1 and Th17 cells, and enhanced anti-inflammatory Treg cells. We speculate that CK2 kinase activity in DCs is indispensable for the initiation and progression of EAE. Specifically, our data indicate that deletion of CK2α reduces the activation of bone marrow-derived DCs, but not macrophages, by suppressing the expression of CD40, TNF-α, iNOS and IL-12 and activation of AKT and p38. Furthermore, we found that activation of DCs is prevented in EAE mice with global CK2α deletion, which correlates with differential regulation of Th1, Th17 and Treg cells. Collectively, our findings establish a novel regulatory role for CK2 kinase in DCs during EAE initiation and progression, which may provide evidence to establish CK2 as a potential novel therapeutic target for MS treatment.

Abstract 108: LMO7, a Negative Feedback Regulator of TGF-beta Signaling and a New Player in Vascular Diseases

Vascular smooth muscle cells (SMC) synthesize extracellular matrix (ECM) that contributes to tissue remodeling following revascularization interventions. The cytokine transforming growth factor-β (TGF-β) is induced upon tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM domain protein LMO7 is a TGF-β target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. We employ carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula (AVF) and cardiac allograft vasculopathy (CAV) samples to assess the role of LMO7 in neointima and fibrosis. We demonstrate that LMO7 is induced post-injury and by TGF-β in SMC in vitro . Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-β signaling, ECM deposition and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and following TGF-β treatment. TGF-β neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-β signaling by inducing expression of Tgfb1 mRNA, TGF-β protein, αv and β3 integrins that promote activation of latent TGF-β, and downstream effectors pSMAD3 and CTGF. Mechanistically, the LMO7 LIM domain interacts with AP-1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting AP-1-dependent TGF-β autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic AVF and CAV samples, and inversely correlates with pSMAD3 in CAV.

Su1033 – Contrasting Effects of Global Versus Tissue-Specific Deletion of Tumor Necrosis Factor Receptor 1(TNFr1) in Murine Colitis

Su1033 – Contrasting Effects of Global Versus Tissue-Specific Deletion of Tumor Necrosis Factor Receptor 1(TNFr1) in Murine Colitis

Foxc2 is essential for podocyte function.

Foxc2 is one of the earliest podocyte markers during glomerular development. To circumvent embryonic lethal effects of global deletion of Foxc2, and to specifically investigate the role of Foxc2 in podocytes, we generated mice with a podocyte‐specific Foxc2 deletion. Mice carrying the homozygous deletion developed early proteinuria which progressed rapidly into end stage kidney failure and death around postnatal day 10. Conditional loss of Foxc2 in podocytes caused typical characteristics of podocyte injury, such as podocyte foot process effacement and podocyte microvillus transformation, probably caused by disruption of the slit diaphragm. These effects were accompanied by a redistribution of several proteins known to be necessary for correct podocyte structure. One target gene that showed reduced glomerular expression was Nrp1, the gene encoding neuropilin 1, a protein that has been linked to diabetic nephropathy and proteinuria. We could show that NRP1 was regulated by Foxc2 in vitro, but podocyte‐specific ablation of Nrp1 in mice did not generate any phenotype in terms of proteinuria, suggesting that the gene might have more important roles in endothelial cells than in podocytes. Taken together, this study highlights a critical role for Foxc2 as an important gene for podocyte function.

Keratinocyte-derived TGF-β is dispensable in skin autoimmunity in mice

Abstract Transforming growth factor beta 1 (TGF-β) required to suppress spontaneous autoimmunity as global deletion of TGF-β resulted in severe multi-organ autoimmunity in mice. Some TGF-β dificient mice showed skin phenotype. However, the effect of TGF-β on the autoimmunity in skin is not clear. In the epidermis of the skin, keratinocytes(KCs) are the major source of TGF-β. To test whether KCs are an obligate source of TGF-β, we generated K14-Cre-ERT2 TGF-βfl/fl mice. Topical application of tamoxifen could result in efficient deletion of TGF-β. We noted increase epidermal thickness but failed to observe evidence of cutaneous inflammation as measured by histology and flow cytometry. Notably, numbers of regulatory T cells were unaffected by the absence of KC-derived TGF-β. Since αvβ6- and αvβ8-integrins (Itgb6 and Itgb8) are principal source of activation of TGF-β in skin and then we generated Itgb6−/− Itgb8ΔKC. Similarly, we did not observe the development of spontaneous inflammation in Itgb6−/− Itgb8ΔKC mice which lack KC-derived TGFβ-activating integrins in the epidermis. From these data we concluded that KC-derived TGF-β and epidermal activation of TGF-β are not required to suppress spontaneous inflammation.

Protein Kinase CK2 Drives Plasma Cell Differentiation

Abstract Casein kinase 2 (CK2) is a serine/threonine protein kinase composed of three subunits: two catalytic subunits, CK2α and CK2α’, and the regulatory subunit CK2β. It is overexpressed and overactive in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphomas, leading to inappropriate activation of NF-κB, JAK/STAT and PI3K/AKT/mTOR signaling, but little is known about how CK2 functions in normal B-cell differentiation. Here, we demonstrate that CK2 mRNA and protein expression is induced upon B-cell activation. Using the small molecule inhibitor CX-4945, which targets the catalytic activity of CK2, a significant inhibition of plasma cell differentiation was observed in vitro under T-cell-independent (TI) or T-cell-dependent (TD) conditions via stimulation with lipopolysaccharide (LPS) or CD40L and interleukin 4 (IL-4) +IL-5, respectively. At the cellular level, we found that inhibition of CK2 kinase activity in B-cells resulted in defective mTORC1 signaling upon stimulation with LPS. These results were confirmed in purified mature B-cells from mice with tamoxifen-induced global deletion of CK2α. These data demonstrate that CK2α is an important regulator of B-cell differentiation. Ongoing experiments with B-cell specific deletion of CK2α will further examine the impact of CK2α on B-cell differentiation in vivo.

Metabolic disease and ABHD6 alter the circulating bis(monoacylglycerol)phosphate profile in mice and humans

Bis(monoacylglycerol)phosphate (BMP) is a phospholipid that is crucial for lipid degradation and sorting in acidic organelles. Genetic and drug-induced lysosomal storage disorders (LSDs) are associated with increased BMP concentrations in tissues and in the circulation. Data on BMP in disorders other than LSDs, however, are scarce, and key enzymes regulating BMP metabolism remain elusive. Here, we demonstrate that common metabolic disorders and the intracellular BMP hydrolase α/β-hydrolase domain-containing 6 (ABHD6) affect BMP metabolism in mice and humans. In mice, dietary lipid overload strongly affects BMP concentration and FA composition in the liver and plasma, similar to what has been observed in LSDs. Notably, distinct changes in the BMP FA profile enable a clear distinction between lipid overload and drug-induced LSDs. Global deletion of ABHD6 increases circulating BMP concentrations but does not cause LSDs. In humans, nonalcoholic fatty liver disease and liver cirrhosis affect the serum BMP FA composition and concentration. Furthermore, we identified a patient with a loss-of-function mutation in the ABHD6 gene, leading to an altered circulating BMP profile. In conclusion, our results suggest that common metabolic diseases and ABHD6 affect BMP metabolism in mice and humans.

PGE2 signaling via the neuronal EP2 receptor increases injury in a model of cerebral ischemia

The inflammatory prostaglandin E2 (PGE2) EP2 receptor is a master suppressor of beneficial microglial function, and myeloid EP2 signaling ablation reduces pathology in models of inflammatory neurodegeneration. Here, we investigated the role of PGE2 EP2 signaling in a model of stroke in which the initial cerebral ischemic event is followed by an extended poststroke inflammatory response. Myeloid lineage cell-specific EP2 knockdown in Cd11bCre;EP2lox/lox mice attenuated brain infiltration of Cd11b+CD45hi macrophages and CD45+Ly6Ghi neutrophils, indicating that inflammatory EP2 signaling participates in the poststroke immune response. Inducible global deletion of the EP2 receptor in adult ROSA26-CreERT2 (ROSACreER);EP2lox/lox mice also reduced brain myeloid cell trafficking but additionally reduced stroke severity, suggesting that nonimmune EP2 receptor-expressing cell types contribute to cerebral injury. EP2 receptor expression was highly induced in neurons in the ischemic hemisphere, and postnatal deletion of the neuronal EP2 receptor in Thy1Cre;EP2lox/lox mice reduced cerebral ischemic injury. These findings diverge from previous studies of congenitally null EP2 receptor mice where a global deletion increases cerebral ischemic injury. Moreover, ROSACreER;EP2lox/lox mice, unlike EP2-/- mice, exhibited normal learning and memory, suggesting a confounding effect from congenital EP2 receptor deletion. Taken together with a precedent that inhibition of EP2 signaling is protective in inflammatory neurodegeneration, these data lend support to translational approaches targeting the EP2 receptor to reduce inflammation and neuronal injury that occur after stroke.

SUN-218 The Role Of Fstl3 In Spermatogenesis: A Transcriptome Analysis

Spermatogenesis in mammals depends on the function of Sertoli cells (SC). The total number of SC present in the testes is determined in the pre-pubertal stages of development. Activin, a TGFβ family ligand, regulates SC proliferation in the early postnatal phase in mice. During this phase immature SC are unable to assemble tight-junctions which are required to form the blood-testis-barrier (BTB) and consequently do not support spermatogenesis. Overexpression of activin beyond pre-pubertal stages results in the disruption of spermatogenesis and results in infertility. Follistatin Like-3 (Fstl3) is a glycoprotein known to bind and inhibit activin. Contrary to overexpression of activin, however, global deletion of Fstl3 in mice results in increased numbers of SC and increased sperm production in older males. Based on this striking observation, in this study, using the global deleted Fstl3 mouse model, we sought to elucidate the function of Fstl3 in testicular development during the transition from pre-pubertal to post-pubertal stages. In testis sections from BrdU treated Fstl3 KO mice, we found that SC number per tubule is maintained from pre-pubertal to post-pubertal stages without a concomitant increase in BrdU positive SC. In contrast, SC numbers in similarly treated WT mice are depleted over the same time period. Stereological analyses further show that although the overall number of SC increases in both genotypes during pre-pubertal stages, increase in SC numbers is significantly higher in Fstl3 KO males. We also found by immunofluorescence that Sycp3 organization appears early (postnatal day 7) in Fstl3 KO mouse testes indicating early meiotic entry. To identify signalling pathways associated with SC proliferation and maturation that are affected by Fstl3 deletion we performed total mRNA sequencing and compared the findings between WT and Fstl3 KO testes of 3 day and 8 weeks mice. Our results show that more than 1000 genes are differentially expressed between WT and KO testes. Pathway analyses reveal that the “Sertoli-to-Sertoli cell Communication” pathway is one of the most relevant enriched canonical pathways in 3 day old Fstl3 KO mice compared to WT. We are currently investigating the mechanisms for early establishment of BTB in Fstl3 KO testes. The results of the study presented suggests that loss of Fstl3 promotes the establishment of SC-SC interactions that support spermatogenesis during the proliferative phase of these cells.

SUN-079 Loss of Endothelial CEACAM1 Induces Atherogenic Fibrous Plaque Formation

Loss of Endothelial CEACAM1 Induces Atherogenic Fibrous Plaque Formation Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1), a substrate of insulin and VEGF receptors, regulates insulin sensitivity by promoting hepatic insulin clearance. Global Ceacam1 gene deletion impaired insulin clearance to cause chronic hyperinsulinemia and peripheral insulin resistance [1]. It also caused spontaneous endothelial dysfunction and hypertension [2] in addition to atherogenic-like plaques in addition to lipid and macrophage deposition, as well as elevated leukocyte rolling and adhesion to the aortic wall in the absence of hypercholesterolemia and hypertriglyceridemia when the mutation was propagated on the C57BL/6 genetic background [3]. Because CEACAM1 promotes endothelial survival, vascular integrity, and pro-angiogenesis, we investigated the cell-autonomous role of endothelial cell CEACAM1 in the pathogenesis of atherosclerosis. To this aim, we generated endothelial cell CEACAM1 knockout mice and backcrossed them with low density lipoprotein receptor deficient mice (ldlr–/– VECad+Cc1fl/fl) prior to feeding them a high cholesterol diet for 3 months and characterizing both of their metabolic and vascular phenotypes. By comparison to ldlr–/– mice, the mutants exhibited insulin sensitivity and moderate hypercholesterolemia. However, they exhibited decreased nitric oxide (NO) bioavailability with a reciprocal increase in plasma endothelin-1 and increased oxidative stress. The mutants also developed fibrotic plaques with elevated macrophage recruitment on the aortic wall without lipid deposition. We postulate that endothelin-1 contributes to fibrosis in these mice. Elevated endothelin-1 levels could stem from increased ras-MAPKinase signaling pathways in response to lower sequestration of Shc and increased coupling of Grb2 to insulin and VEGR in the absence of Ceacam1. Moreover, in the absence of Ceacam1, binding of Shp2 phosphatase to IRS1 increases to reduce IRS-1/Akt/eNOS activity and subsequently, lower NO production. Thus, the ldlr–/– VECad+Cc1fl/fl mouse can provide an in vivo system to delineate the endothelial cell-specific CEACAM1 effect on fibrosis independent of systemic risk factors.

N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

HSD17B12 Is Essential for the Metabolic Homeostasis in Adult Mice

Hydroxysteroid 17‐beta dehydrogenase (HSD17B) enzymes have an important role in the regulation of sex steroid metabolism by catalyzing the conversion between 17‐keto and 17‐hydroxysteroids. In addition, some HSD17B enzymes are involved in lipid metabolism. Our data together with other previous studies suggest that HSD17B12 is involved in the elongation of very long chain fatty acids (VLCFAs), presumably in producing arachidonic acid. The enzyme presents with universal expression pattern both in human and mouse. Our previous studies have shown that the global knockout of Hsd17b12 gene in mice is embryonic lethal, which is likely due to its pivotal role in the elongation of VLCFAs during development. The aim of the present study was to determine the role of HSD17B12 at adulthood. We, thus, generated conditional Hsd17b12 knockout mice with exon 2 flanked by loxP sites. The gene was globally deleted by crossing the floxed mice with those expressing the tamoxifen‐inducible Cre recombinase in the Rosa26 locus (HSD17B12cKOrosa26 mice) and by providing tamoxifen treatment at the age of eight weeks. Interestingly, inducing the global gene deletion led to 20% reduction in body weight, associated with drastically reduced amount of both white and brown adipose tissue. Our results with indirect calorimetry showed that food intake and water consumption were dramatically reduced (44% and 65%, respectively) in HSD17B12cKOrosa26 mice, although the hypothalamic regulation of feeding behavior was intact. The loss of appetite might be a consequence of general indisposition of the animals, however, no differences in motor activity was observed between KO and control mice. To study whether the strong metabolic phenotype is caused by an altered adipose tissue function, we then generated an inducible, adipocyte‐specific Hsd17b12 KO model (HSD17B12cKOadipoq) by crossbreeding the floxed mice with those expressing the tamoxifen‐inducible Cre recombinase under adiponectin promoter (Adipoq‐cre/ERT2 mice). Surprisingly, the HSD17B12cKOadipoq mice were viable, presented with normal body weight, and were free of any obvious abnormalities. Because of the high hepatic expression of HSD17B12 in both human and mouse, we generated a liver‐specific knockout mouse by crossbreeding the floxed mouse strain with Albumin‐Cre mouse strain. These mice were viable but presented with lipid accumulation in the hepatocytes at a young age (2 months). The results indicate that HSD17B12 is essential for the metabolic homeostasis in adult mice, while the vital role of the enzyme in metabolic regulation is not due to the high HSD17B12 activity in the adipocytes or hepatocytes. Moreover, the hepatocyte‐specific deletion of Hsd17b12 leads to liver steatosis in mice.Support or Funding InformationThis research was funded by Sigrid Jusélius foundation, Academy of Finland and Drug Research Doctoral Program at the University of Turku.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abrogation of transforming growth factor-β-induced tissue fibrosis in mice with a global genetic deletion of Nox4

Excessive connective tissue deposition in skin and various internal organs is characteristic of systemic sclerosis (SSc). The profibrotic growth factor TGF-β plays a crucial role in SSc pathogenesis. The expression of NADPH oxidase 4 (NOX4), a critical mediator of oxidative stress, is potently stimulated by TGF-β. Here, we evaluated the effect of NOX4 on the development of TGF-β-induced tissue fibrosis. C57BL6/J control mice and Nox4 knockout mice were implanted subcutaneously with osmotic pumps containing either saline or 2.5 µg TGF-β1. After 28 days, skin and lung samples were isolated for histopathologic analysis, measurement of hydroxyproline content and gene expression analysis. Histopathology of skin and lungs from normal C57BL6/J mice treated with TGF-β1 showed profound dermal fibrosis and peribronchial and diffuse interstitial lung fibrosis. In contrast, TGF-β-treated Nox4 knockout mice showed normal skin and lung histology. Hydroxyproline levels in TGF-β-treated C57BL6/J mice skin and lungs demonstrated significant increases, however, hydroxyproline content of TGF-β-treated Nox4 knockout mice tissues was not changed. Expression of various profibrotic and fibrosis-associated genes was upregulated in skin and lungs of TGF-β1-treated C57BL6/J mice but was not significantly changed in TGF-β1-treated Nox4 knockout mice. The induction of skin and lung tissue fibrosis by TGF-β1 parenteral administration in mice was abrogated by the genetic deletion of Nox4 confirming that NOX4 is an essential mediator of the profibrotic effects of TGF-β. These results suggest Nox4 inhibition as a potential therapeutic target for SSc and other fibroproliferative disorders.

Platelet-type 12-lipoxygenase deletion provokes a compensatory 12/15-lipoxygenase increase that exacerbates oxidative stress in mouse islet β cells

In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12-/- mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15-/- mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress.

Cardiomyocyte‐Specific EGFR Deletion Differentially Alters Cardiac Contractility and Remodeling

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissues and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well‐explored. Using mice with floxed EGFR alleles crossed with αMHC‐Cre mice, we have generated a cardiomyocyte‐specific constitutive EGFR knockout mouse model (CM‐EGFR‐KO) to address its role in the heart. Compared to their wild‐type (WT) littermate controls, CM‐EGFR‐KO mice displayed age‐related development of cardiac contractile dysfunction, marked by impaired diastolic relaxation and Ca2+ handling at the individual myocyte level, as monitored via echocardiographic, hemodynamic, myocyte contractility and Ca2+ transient analyses. RNASeq analysis of CM‐EGFR‐KO versus WT littermate left ventricular samples revealed temporally‐dependent alterations in the expression of a number of myofilament, Ca2+ handling and fetal genes. The contractile defect initially occurs between 7 and 9 weeks of age and is maintained over time without overt cardiac hypertrophic or fibrotic remodeling until 10 months of age, by which time the mice ultimately succumb to severe heart failure and reduced lifespan. Chronic administration of the β‐adrenergic receptor (βAR) agonist isoproterenol effectively compensated for the contractile dysfunction in CM‐EGFR‐KO mice, an effect that was reversible upon cessation of infusion. Notably, βAR‐mediated cardiomyocyte hypertrophy was attenuated in CM‐EGFR‐KO mice compared to WT littermate controls. Treatment of 16 week‐old CM‐EGFR‐KO mice with AAV9‐cTnT‐EGFR permanently rescued cardiomyocyte‐specific EGFR expression and contractile function, suggesting that the contractile dysfunction phenotype observed in these mice is not secondary to aberrant cardiac development, but is specific to EGFR‐dependent regulation of contractile signaling.Support or Funding InformationNIH grant HL105414 to DGTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Post‐Natal Growth and Liver Immune Homeostasis are Post‐Transcriptionally Regulated by Tristetraprolin Family of RNA Binding Proteins

Tristetraprolin family of RNA binding proteins, including tristetraprolin (TTP), zinc finger protein 36 like 1 (ZFP36L1), and zinc finger protein 36 like 2 (ZFP36L2) regulate mRNA levels by binding to AU‐rich elements on the 3′ untranslated regions of specific mRNAs and enhancing their turnover. While the three proteins behave similarly under biochemical conditions, they appear to have diverse physiological functions. This is apparent from the global deletion of the three proteins in mice. While the global loss of TTP results in systemic inflammatory syndrome, global loss of ZFP36L1 is embryonic lethal, and the global loss of ZFP36L2 results in early post‐natal mortality. Here, we sought to understand the physiological functions of the three proteins in liver by conditional liver specific deletion using Cre‐loxP technology. While the independent deletion of the three proteins did not result in any aberrant phenotype, simultaneous deletion resulted in post‐natal growth defect, chronic active portal hepatitis, and bile duct hyperplasia. Transcriptomic analysis showed small numbers of gene expression changes in the liver of ZFP36L1 knockout, no changes in the livers of TTP and ZFP36L2 knockout, and many changes in the liver of mice deleted of the three proteins simultaneously. The changes included perturbation of genes that regulate post‐natal growth and several inflammatory mediators, some of which are novel potential liver‐specific targets of TTP family proteins. Interestingly, one allele of either ZFP36L1 or ZFP36L2 was sufficient to protect the mice from developing this phenotype. However, one allele of TTP was only partially protective. In summary, our results suggest that post‐transcriptional regulation of gene expression plays an important role in post‐natal growth and liver immune homeostasis and that TTP family proteins regulate these physiological functions in both specific and redundant manner.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arginase 2 Overexpression Aggravates Ischemic Injury in Retinal Vascular Endothelial Cells

ObjectiveOur group has previously shown that degeneration of the retinal microvasculature after ischemia/reperfusion (I/R) injury is mediated through increased expression of the mitochondrial isoform of arginase, arginase 2 (A2). In contrast, increased expression of the cytosolic isoform, arginase 1 (A1) is involved in impairment of endothelial cell (EC)‐dependent vasorelaxation. We have shown that global deletion of A2 limits I/R‐induced vascular degeneration whereas EC‐specific deletion of A1 prevents diabetes‐induced EC dysfunction. However, the specific involvement of EC arginase expression in I/R‐induced vascular EC injury is as yet unknown. Now we have investigated the specific impact of increasing EC expression of A1 or A2 on EC survival in relation to mitochondrial function by studies using an in vitro model of I/R injury.MethodsStudies were performed using bovine retinal ECs transduced with or without adenoviral vectors to overexpress A1 or A2. I/R was modeled by subjecting the cultures to oxygen glucose deprivation (OGD) for 5 hours followed by reperfusion for 1 hour (OGD/R). Some cultures were treated with or without the specific but non‐selective inhibitor of arginase ABH (amino‐2‐borono‐6‐hexanoic acid). Cell extracts were collected for western blot. Media were collected to evaluate cell death using lactate dehydrogenase (LDH) release assay. Seahorse XF analyzer was used to evaluate mitochondrial function.ResultsStudies using normal ECs exposed to OGD/R in the presence or absence of ABH showed that inhibiting arginase protects against mitochondrial injury as shown by decreases in mitochondrial fragmentation and reduced expression of the mitochondrial fission marker Drp1 (dynamin related protein 1). Western blot analysis showed that OGD/R promoted a significant increase in A2 expression whereas A1 expression was unchanged. Seahorse analysis of the oxygen consumption rate (OCR), under normoxic conditions, showed that A2 overexpression significantly decreased basal respiration and ATP production indicating mitochondrial dysfunction. OGD/R insult in non‐transduced ECs caused mitochondrial dysfunction as demonstrated by decreased basal respiration, reduced ATP production and decreased spare respiratory capacity. A2 overexpression significantly aggravated the OGD/R‐induced mitochondrial dysfunction as shown by further decreases in basal respiration and ATP production. Overexpression of A1 had no effect on these parameters. A2 overexpression also increased phosphorylation of the stress kinase p38 mitogen‐activated protein kinase (p38‐MAPK) and increased release of LDH as compared to OGD/R alone indicating increases in cell stress and cell death. Overexpression of A1 had no effect on either measure.ConclusionsThe present study is the first to show that A2 could be playing a role in promoting OGD/R‐induced cell death through mediating mitochondrial dysfunction.Support or Funding InformationR01‐EY11766, VA grant BX001233, Culver Vision Discovery Institute at Augusta UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.