Perivascular adipose tissue-derived extracellular vesicle miR-221-3p mediates vascular remodeling.

Adipose tissue-secreted extracellular vesicles (EVs) containing microRNAs (miRNAs) convey intercellular message signaling. The biogenesis of EV-miRNAs from perivascular adipose tissue (PVAT) and their roles in intercellular communication in response to obesity-associated inflammation have not yet been fully explored. By feeding mice a high-fat diet for 16 wk, we established obesity-associated, chronic low-grade inflammation in PVAT, characterized as hypertrophy of perivascular adipocytes, decreased adipogenesis, and proinflammatory macrophage infiltration. We show that PVAT-derived EVs and their encapsulated miRNAs can be taken up into vascular smooth muscle cells (VSMCs) in vivo and in vitro. miR-221-3p is one of the highly enriched miRNAs in obese PVAT and PVAT-derived EVs. Transfer and direct overexpression of miR-221-3p dramatically enhances VSMC proliferation and migration. Peroxisome proliferator-activated receptor γ coactivator 1α is identified as a miR-221-3p target in VSMC phenotypic modulation. Obese mice secrete abundant miRNA-containing EVs, evoking inflammatory responses in PVAT and vascular phenotypic switching in abdominal aorta of lean mice. Local delivery of miR-221-3p mimic in femoral artery causes vascular dysfunction by suppressing the contractile genes in the arterial wall. Our findings provide an EV-miR-221-3p-mediated mechanism by which PVAT triggers an early-stage vascular remodeling in the context of obesity-associated inflammation.-Li, X., Ballantyne, L. L., Yu, Y., Funk, C. D. Perivascular adipose tissue-derived extracellular vesicle miR-221-3p mediates vascular remodeling.

Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-synthetic capacity under basal conditions.

The distinct functions of each cyclooxygenase (COX) isoform in renal homeostasis have been the subject of intense investigation for many years. We took the novel approach of using 3 characterized mouse lines, where the prostaglandin (PG)-endoperoxide synthase genes 1 and 2 ( Ptgs1 and Ptgs2) substitute for one another to delineate distinct roles and the potential for COX isoform substitution. Flipped Ptgs genes generate a reversed COX-expression pattern in the kidney, where the knockin COX-2 is highly expressed. Normal nephrogenesis was sustained in all 3 strains at the postnatal stage d 8 (P8). Knockin COX-1 can temporally restore renal function and delay but not prevent renal pathology consequent to COX-2 deletion. Loss of COX-2 in adult COX-1 > COX-2 mice results in severe nephropathy, which leads to impaired renal function. These defects are partially rescued by the knockin COX-2 in Reversa mice, whereas COX-2 can compensate for the loss of COX-1 in COX-2 > COX-1 mice. Intriguingly, the highly expressed knockin COX-2 enzyme barely makes any PGs or thromboxane in neonatal P8 or adult mice, demonstrating that prostanoid biosynthesis requires native COX-1 and cannot be rescued by the knockin COX-2. In summary, the 2 COX isoforms can preferentially compensate for some renal functions, which appears to be independent of the PG-synthetic capacity.-Li, X., Mazaleuskaya, L. L., Ballantyne, L. L., Meng, H., FitzGerald, G. A., Funk, C. D. Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-synthetic capacity under basal conditions.

Genomic and lipidomic analyses differentiate the compensatory roles of two COX isoforms during systemic inflammation in mice.

Both cyclooxygenase (COX)-1 and COX-2, encoded by Ptgs1 and Ptgs2, function coordinately during inflammation. But the relative contributions and compensations of COX-1 and COX-2 to inflammatory responses remain unanswered. We used three engineered mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another to discriminate the distinct roles and interchangeability of COX isoforms during systemic inflammation. In macrophages, kidneys, and lungs, "flipped" Ptgs genes generate a "reversed" COX expression pattern, where the knock-in COX-2 is expressed constitutively and the knock-in COX-1 is lipopolysaccharide inducible. A panel of eicosanoids detected in serum and kidney demonstrates that prostaglandin (PG) biosynthesis requires native COX-1 and cannot be rescued by the knock-in COX-2. Our data further reveal preferential compensation of COX isoforms for prostanoid production in macrophages and throughout the body, as reflected by urinary PG metabolites. NanoString analysis indicates that inflammatory networks can be maintained by isoform substitution in inflamed macrophages. However, COX-1>COX-2 macrophages show reduced activation of inflammatory signaling pathways, indicating that COX-1 may be replaced by COX-2 within this complex milieu, but not vice versa. Collectively, each COX isoform plays a distinct role subject to subcellular environment and tissue/cell-specific conditions, leading to subtle compensatory differences during systemic inflammation.

Flipping the cyclooxygenase (Ptgs) genes reveals isoform-specific compensatory functions.

Two prostaglandin (PG) H synthases encoded by Ptgs genes, colloquially known as cyclooxygenase (COX)-1 and COX-2, catalyze the formation of PG endoperoxide H2, the precursor of the major prostanoids. To address the functional interchangeability of these two isoforms and their distinct roles, we have generated COX-2>COX-1 mice whereby Ptgs2 is knocked in to the Ptgs1 locus. We then "flipped" Ptgs genes to successfully create the Reversa mouse strain, where knock-in COX-2 is expressed constitutively and knock-in COX-1 is lipopolysaccharide (LPS) inducible. In macrophages, flipping the two Ptgs genes has no obvious impact on COX protein subcellular localization. COX-1 was shown to compensate for PG synthesis at high concentrations of substrate, whereas elevated LPS-induced PG production was only observed for cells expressing endogenous COX-2. Differential tissue-specific patterns of expression of the knock-in proteins were evident. Thus, platelets from COX-2>COX-1 and Reversa mice failed to express knock-in COX-2 and, therefore, thromboxane (Tx) production in vitro and urinary Tx metabolite formation in COX-2>COX-1 and Reversa mice in vivo were substantially decreased relative to WT and COX-1>COX-2 mice. Manipulation of COXs revealed isoform-specific compensatory functions and variable degrees of interchangeability for PG biosynthesis in cells/tissues.

Multiple-site activation of the cysteinyl leukotriene receptor 2 is required for exacerbation of ischemia/reperfusion injury.

OBJECTIVE: Transgenic overexpression of the human cysteinyl leukotriene receptor 2 (CysLT2R) in murine endothelium exacerbates vascular permeability and ischemia/reperfusion injury. Here, we explore the underlying mechanisms of CysLT2R activation-mediated inflammation and delineate the relative contributions of endogenous murine CysLT2R and the transgene-derived receptor. APPROACH AND RESULTS: We created a novel mouse with only endothelial-expressed CysLT2R (endothelium-targeted overexpression mice [EC]/CysLT2R-knockout mice [KO]) by crossing EC with KO to dissect the role of endothelial CysLT2R in tissue injury. Surprisingly, we discovered that damage in EC/KO mice was not elevated (24% versus 47% EC) after ischemia/reperfusion. We examined vascular permeability and leukocyte recruitment/rolling responses in the cremaster vasculature after cysteinyl leukotriene (cysLT) stimulation. Mice possessing transgenic endothelial CysLT2R overexpression, whether EC or EC/KO, when stimulated with cysLTs, exhibited vascular hyperpermeability, declining leukocyte flux, and a transient increase in slow-rolling leukocyte fraction. Mice lacking endogenous CysLT2R (both KO [20 ± 3 cells/min] EC/KO [24 ± 3]) showed lower-rolling leukocyte flux versus wild-type (38 ± 6) and EC (35 ± 6) mice under unstimulated conditions. EC/KO mice differed from EC counterparts in that vascular hyperpermeability was not present in the absence of exogenous cysLTs. CONCLUSIONS: These results indicate that endothelial and nonendothelial CysLT2R niches have separate roles in mediating inflammatory responses. Endothelial receptor activation results in increased vascular permeability and leukocyte slow-rolling, facilitating leukocyte transmigration. Nonendothelial receptors, likely located on resident/circulating leukocytes, facilitate endothelial receptor activation and leukocyte transit. Activation of both receptor populations is required for injury exacerbation.

MyD88-dependent Toll-like receptor 2 signaling modulates macrophage activation on lysate-adsorbed Teflon™ AF surfaces in an in vitro biomaterial host response model.

The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAW-Blue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1ß, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMP-related proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway.

The extended effect of adsorbed damage-associated molecular patterns and Toll-like receptor 2 signaling on macrophage-material interactions.

Implanted biomaterials elicit an immune-mediated foreign body reaction (FBR) that results in the fibrous encapsulation of the implant and can critically impact the performance of some implants. Consequently, understanding the molecular mechanisms that underpin cell-materials interactions that initiate biomaterial-induced inflammation and fibrosis is critical to improving the performance of biomaterial implants negatively impacted by the FBR. Damage-associated molecular patterns (DAMPs) are endogenous mediators of inflammation that are released upon tissue injury and induce sterile inflammation via Toll-like receptors (TLRs). However, the prevalence of DAMPs within the adsorbed protein layer on material surfaces and their role mediating cell-material interactions is unclear. Previously, our group demonstrated that molecules in fibroblast lysates adsorbed to various biomaterials and induced a potent TLR2-dependent inflammatory response in macrophages at 24 h. In this study, we examined the extended response of RAW-Blue reporter macrophages on lysate or serum-adsorbed Teflon™ AF surfaces to understand the potential role of adsorbed DAMPs in macrophage-material interactions at later time points. Lysate-conditioned surfaces maintained increased nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) transcription factor activity and increased expression Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES/CCL5) at 72 h and 120 h, compared to FBS-conditioned surfaces. In contrast, monocyte chemoattractant protein 1 (MCP-1/CCL2) was only elevated at 72 h in lysate conditions. Transforming growth factor beta 1 (TGF-ß1) secretion was significantly increased on lysate-conditioned surfaces, while conditioned media from macrophages on lysate-conditioned surfaces induced alpha smooth muscle actin (αSMA) expression in 3T3 fibroblasts. TLR2 neutralizing antibody treatment significantly decreased NF-κB/AP-1 activity and attenuated TGF-ß1 expression at both time points, and MCP-1 and RANTES at 72 h. Finally, multinucleated cells were observed on lysate-conditioned surfaces at 72 h, indicating adsorbed DAMPs induced a fusion permissive environment for adherent macrophages. This study demonstrates that adsorbed DAMPs continue to influence macrophage-material responses beyond the initial 24-h period and maintain a pro-inflammatory and fibrotic response that models aspects of the early FBR. Furthermore, the transient inhibition of TLR2 continued to exert an effect at these later time points, suggesting TLR2 may be a target for therapeutic interventions in FBR.

A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

Arginase-1 deficiency in neural cells does not contribute to neurodevelopment or functional outcomes after sciatic nerve injury.

Arginase-1 (Arg1) is an enzyme controlling the final step of the urea cycle, with highest expression in the liver and lower expression in the lungs, pancreas, kidney, and some blood cells. Arg1 deficiency is an inherited urea cycle disorder presenting with neurological dysfunction including spastic diplegia, intellectual and growth retardation, and encephalopathy. The contribution of Arg1 expression in the central and peripheral nervous system to the development of neurological phenotypes remains largely unknown. Previous studies have shown prominent arginase-1 expression in the nervous system and post-peripheral nerve injury in mice, but very low levels in the naïve state. To investigate neurobiological roles of Arg1, we created a conditional neural (n)Arg1 knockout (KO) mouse strain, with expression eliminated in neuronal and glial precursors, and compared them to littermate controls. Long-term analysis did not reveal any major differences in blood amino acid levels, body weight, or stride gait cycle from 8 to 26-weeks of age. Brain structure measured by magnetic resonance imaging at 16-weeks of age observed only a significant decrease in the volume of the mammillary bodies. We also assessed whether nArg1, which is expressed by sensory neurons after injury, may play a role in regeneration following sciatic nerve crush. Only subtle differences were observed in locomotor and sensory recovery between nArg1 KO and control mice. These results suggest that arginase-1 expression in central and peripheral neural cells does not contribute substantially to the phenotypes of this urea cycle disorder, nor is it likely crucial for post-injury regeneration in this mouse model.

Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice.

Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC) technology. Here, we report transcription activator-like effector nuclease (TALEN)-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs) in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013) tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

Isoform-Specific Compensation of Cyclooxygenase (Ptgs) Genes during Implantation and Late-Stage Pregnancy.

The participation of cyclooxygenase (COX) in embryo implantation and parturition has been studied extensively. However, the distinct role of the two COX isoforms in these processes still remains unclear. Using three characterized mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another, this study focused on the reproductive significance of their distinct roles and potential biological substitution. In both non-gravid and gravid uteri, the knock-in COX-2 is expressed constitutively, whereas the knock-in COX-1 is slightly induced in early implantation. The delayed onset of parturition previously found in COX-1 null mice was corrected by COX-2 exchange in COX-2>COX-1 mice, with normal term pregnancy, gestation length and litter size. In contrast, loss of native COX-2 in COX-1>COX-2 mice resulted in severely impaired reproductive functions. Knock-in COX-1 failed to substitute for the loss of COX-2 in COX-1>COX-2 mice during implantation, indicating that COX-1 may be replaced by COX-2, but not vice versa. A panel of prostaglandins detected in uterus and ovary demonstrates that prostaglandin biosynthesis preferentially depends on native COX-1, but not COX-2. More interestingly, preferential compensations by the COX isoforms were sustained despite weak dependency on their role in prostaglandin biosynthesis in the uterus and ovary.

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency.

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of "repaired" arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

Liver-specific knockout of arginase-1 leads to a profound phenotype similar to inducible whole body arginase-1 deficiency.

Arginase-1 (Arg1) converts arginine to urea and ornithine in the distal step of the urea cycle in liver. We previously generated a tamoxifen-inducible Arg1 deficient mouse model (Arg1-Cre) that disrupts Arg1 expression throughout the whole body and leads to lethality ≈ 2 weeks after gene disruption. Here, we evaluate if liver-selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of gene delivery or hepatocyte transplantation to rescue the phenotype. Liver-selective Arg1 deletion was induced by using an adeno-associated viral (AAV)-thyroxine binding globulin (TBG) promoter-Cre recombinase vector administered to Arg1 "floxed" mice; Arg1fl/fl ). An AAV vector expressing an Arg1-enhanced green fluorescent protein (Arg1-eGFP) transgene was used for gene delivery, while intrasplenic injection of wild-type (WT) C57BL/6 hepatocytes after partial hepatectomy was used for cell delivery to "rescue" tamoxifen-treated Arg1-Cre mice. The results indicate that liver-selective loss of Arg1 (> 90% deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality ≈ 3 weeks after Cre-induced gene disruption. Delivery of Arg1-eGFP AAV rescues more than half of Arg1 global knockout male mice (survival > 4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels observed in WT animals. Significant Arg1 enzyme activity from engrafted WT hepatocytes into knockout livers can be achieved but not sufficient for rescuing the lethal phenotype. This raises a conundrum relating to liver-specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, gene and cell-directed therapies suggest that rescue of extra-hepatic Arg1 expression may also be necessary for disease correction. Further studies are needed in order to illuminate the detailed mechanisms for pathogenesis of Arg1-deficiency.

Strategies to rescue the consequences of inducible arginase-1 deficiency in mice.

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken ß-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.

Endogenously generated omega-3 fatty acids attenuate vascular inflammation and neointimal hyperplasia by interaction with free fatty acid receptor 4 in mice.

BACKGROUND: Omega-3 polyunsaturated fatty acids (ω3 PUFAs) suppress inflammation through activation of free fatty acid receptor 4 (FFAR4), but this pathway has not been explored in the context of cardiovascular disease. We aimed to elucidate the involvement of FFAR4 activation by ω3 PUFAs in the process of vascular inflammation and neointimal hyperplasia in mice. METHODS AND RESULTS: We used mice with disruption of FFAR4 (Ffar4(-/-)), along with a strain that synthesizes high levels of ω3 PUFAs (fat-1) and a group of crossed mice (Ffar4(-/-)/fat-1), to elucidate the role of FFAR4 in vascular dysfunction using acute and chronic thrombosis/vascular remodeling models. The presence of FFAR4 in vascular-associated cells including perivascular adipocytes and macrophages, but not platelets, was demonstrated. ω3 PUFAs endogenously generated in fat-1 mice (n=9), but not in compound Ffar4(-/-)/fat-1 mice (n=9), attenuated femoral arterial thrombosis induced by FeCl3. Neointimal hyperplasia and vascular inflammation in the common carotid artery were significantly curtailed 4 weeks after FeCl3 injury in fat-1 mice (n=6). This included greater luminal diameter and enhanced blood flow, reduced intima:media ratio, and diminished macrophage infiltration in the vasculature and perivascular adipose tissue compared with control mice. These effects were attenuated in the Ffar4(-/-)/fat-1 mice. CONCLUSIONS: These results indicate that ω3 PUFAs mitigate vascular inflammation, arterial thrombus formation, and neointimal hyperplasia by interaction with FFAR4 in mice. Moreover, the ω3 PUFA-FFAR4 pathway decreases inflammatory responses with dampened macrophage transmigration and infiltration.

Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism.

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing "floxed" Arg1 mice with CreER(T2) mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.