Cannabinoid receptor 2 signaling does not modulate atherogenesis in mice

Background

Strong evidence supports a protective role of the cannabinoid receptor 2 (CB₂) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB₂ receptor in Murine atherogenesis.

Methods and Findings

Low density lipoprotein receptor-deficient (LDLR^−/−) mice subjected to intraperitoneal injections of the selective CB₂ receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB₂ activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB₂ ^−/−/LDLR^−/− mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB₂ ^+/+/LDLR^−/− controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-illicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB₂ receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro.

Conclusion

Our study demonstrates that both activation and deletion of the CB₂ receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB₂ in other inflammatory processes. However, in the context of atherosclerosis, CB₂ does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque.     

Npp1 promotes atherosclerosis in ApoE knockout mice

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.     

A mechanism for the impaired IFN-gamma production in C-C chemokine receptor 2 (CCR2) knockout mice: role of CCR2 in linking the innate and adaptive immune responses

We have recently shown that mice with a targeted disruption of CCR2, the receptor for monocyte chemoattractant protein-1, have markedly impaired recruitment of macrophages to sites of inflammation. An unexpected finding in the CCR2(-/-) mice was a dramatic decrease in the production of IFN-gamma after challenge with purified protein derivative of Mycobacterium bovis. In this study, we have investigated the mechanism of this cytokine production defect. In vitro, direct activation of splenocytes with CD3/CD28 Abs failed to reveal any differences in IFN-gamma production between CCR2(+/+) and CCR2(-/-) mice. However, after immunization, the number of Ag-specific, IFN-gamma-producing cells in the draining lymph nodes was decreased by 70% in the CCR2(-/-) mice, suggesting an in vivo trafficking defect. Direct measurement of cell trafficking with fluorescently labeled CFA revealed a marked decrease in the number of monocytes/macrophages migrating to the site of immunization and to the draining lymph nodes in the CCR2(-/-) mice. The data suggest that impaired trafficking of APCs in the CCR2(-/-) mice contributes to the defect in IFN-gamma production. These data support the idea that CCR2-positive monocytes/macrophages are critical in linking the innate and adaptive immune responses.     

Suppression of atherogenesis by delivery of TGFbeta1ACT using adeno-associated virus type 2 in LDLR knockout mice

TGFbeta(1) deficiency has been attributed to the development of atherosclerosis. There is, however, little direct evidence for this concept. To examine this hypothesis, low-density lipoprotein receptor knockout (LDLR(-/-)) mice were injected via tail vein with recombinant adeno-associated virus type 2 (rAAV) carrying a bioactive TGFbeta(1) mutant (AAV/TGFbeta1ACT, n=10) or granulocyte-macrophage-colony stimulating factor (AAV/GM-CSF, n=10, a negative control) or saline (n=9, control), and then put on a high cholesterol diet. At 18 weeks, blood lipids were found to be similarly elevated in all LDLR(-/-) mice. TGFbeta1ACT and GM-CSF (DNA, mRNA, and protein) were highly expressed in the tissues of mice given TGFbeta1ACT or AAV/GM-CSF, respectively, showing sustained transfection following gene delivery by the systemic route. Saline-treated and AAV/GM-CSF-treated LDLR(-/-) mice showed extensive areas of atherosclerotic lesion formation. There was evidence of intense oxidative stress (nitrotyrosine staining), inflammation (CD68 staining), and expression of adhesion molecules and the ox-LDL receptor LOX-1 (gene array analysis) in the atherosclerotic tissues. Importantly, atherosclerotic lesion formation was markedly inhibited in the LDLR(-/-) mice given AAV/TGFbeta1ACT. Expression of adhesion molecules and LOX-1, oxidative stress, and inflammatory response all were inhibited in the mice given AAV/TGFbeta1ACT (P<0.05 vs. saline-treated or GM-CSF-treated LDLR(-/-) mice). These data for the first time demonstrate that systemic delivery of TGFbeta1ACT gene via AAV can inhibit formation of atherosclerotic lesions, possibly via anti-inflammatory and anti-oxidant mechanisms. These findings suggest a novel view of TGFbeta(1) in atherogenesis and a potential new gene therapy for treatment of atherosclerosis.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

The chemokine receptor CCR2 is not required for successful initiation of labor in mice

Chemokine-driven neutrophil and monocyte recruitment into the uterus and cervix has been proposed to initiate labor. Chemokines that bind CXCR2 direct neutrophil migration and are induced during labor in humans. The chemokine CCL2, induced in the uterus by endocrine and mechanical signals, has been proposed to drive CCR2-dependent monocyte homing to the uterus to contribute to the initiation of labor. However, no direct evidence indicates that chemokines or their receptors play indispensable roles in labor-associated inflammation, and the impact of leukocyte infiltration on labor is unclear. Here, we have quantified expression of the principal monocyte- and neutrophil-attracting chemokines in the uteri of term pregnant (Day 18) and laboring wild-type mice. None of the neutrophil attractants we assayed were up-regulated with labor. Strikingly, however, Ccl2 was markedly increased, and this was concomitant with increased expression of Ccr2, the myeloid marker Itgam (also known as Cd11b), the monocyte/macrophage marker Emr1 (also known as F4/80). Moreover, in CCR2-deficient mice, this labor-associated increase in Itgam and Emr1 was not seen, consistent with the monocyte-trafficking defects that exist in these animals. Nonetheless, laboring CCR2-deficient and wild-type uteri showed similarly enhanced expression of the myometrial activation markers Gja1 and Oxtr (commonly known as connexin 43 and oxytocin receptor, respectively), and CCR2-deficient mice had gestation lengths, litter sizes, and fetal and placental weights no different from those of their wild-type counterparts. Thus, whereas labor is associated with an inflammatory response in gestational tissues, CCR2-dependent leukocyte recruitment into the mouse uterus is dispensable for the initiation of successful labor.     

Lead identification of benzimidazolone and azabenzimidazolone arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists

A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. Hits were required to be more potent than an existing indazole series, have better physicochemical properties (c log P <3.5, chrom log D_7.4 <5.3 and CLND solubility >116 μg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however, these analogues were inactive in the whole blood assay (pA₂ <5). Azabenzimidazolone analogues were all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight (389) and chrom log D_7.4 (2.4), high LE (0.43), and solubility (152 μg/mL). In addition, 38 had human serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.     

Sensorimotor development in neonatal progesterone receptor knockout mice

Early exposure to steroid hormones can permanently and dramatically alter neural development. This is best understood in the organizational effects of hormones during development of brain regions involved in reproductive behaviors or neuroendocrine function. However, recent evidence strongly suggests that steroid hormones play a vital role in shaping brain regions involved in cognitive behavior such as the cerebral cortex. The most abundantly expressed steroid hormone receptor in the developing rodent cortex is the progesterone receptor (PR). In the rat, PR is initially expressed in the developmentally‐critical subplate at E18, and subsequently in laminas V and II/III through the first three postnatal weeks (Quadros et al. [2007] J Comp Neurol 504:42–56; Lopez & Wagner [2009]: J Comp Neurol 512:124–139), coinciding with significant periods of dendritic maturation, the arrival of afferents and synaptogenesis. In the present study, we investigated PR expression in the neonatal mouse somatosensory cortex. Additionally, to investigate the potential role of PR in developing cortex, we examined sensorimotor function in the first two postnatal weeks in PR knockout mice and their wildtype (WT) and heterozygous (HZ) counterparts. While the three genotypes were similar in most regards, PRKO and HZ mice lost the rooting reflex 2–3 days earlier than WT mice. These studies represent the first developmental behavioral assessment of PRKO mice and suggest PR expression may play an important role in the maturation of cortical connectivity and sensorimotor integration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 16–24, 2014     

Cytomegalovirus infection aggravates atherogenesis in apoE knockout mice by both local and systemic immune activation

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.     

Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P(1-5)) to cardiovascular homeostasis. We used S1P(2) receptor knockout mice (S1P(2)(-/-)) to evaluate the role of S1P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.     

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice

Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.     

The value of apolipoprotein E knockout mice for studying the effects of dietary fat and cholesterol on atherogenesis

The ability of the apolipoprotein E-deficient mouse to develop spontaneous atherosclerosis, which resembles the human process, is an excellent model in which to assess the impact of dietary factors. This review discusses the role of several nutrients in the development of atherosclerosis and the mechanisms through which they act.     

Potent and selective CC-chemokine receptor-2 (CCR2) antagonists as a potential treatment for asthma

A number of compounds bearing a quaternary ammonium moiety were found to be antagonists with nanomolar binding affinity for the chemokine receptor-2. The structure-activity relationships in the series are described herein along with some detailed characterization of the interesting compounds.     

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.     

The cause of infertility of male c-ros tyrosine kinase receptor knockout mice

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46-86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 +/- 119 free, 371 +/- 70 loosely, and 122 +/- 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.     

Hematopoietic CC-chemokine receptor 2 (CCR2) competent cells are protective for the cognitive impairments and amyloid pathology in a transgenic mouse model of Alzheimer's disease

Monocytes emigrate from bone marrow, can infiltrate into brain, differentiate into microglia and clear amyloid β (Aβ) from the brain of mouse models of Alzheimer’s disease (AD). Here we show that these mechanisms specifically require CC-chemokine receptor 2 (CCR2) expression in bone marrow cells (BMCs). Disease progression was exacerbated in APP_Swe/PS1 mice (transgenic mice expressing a chimeric amyloid precursor protein [APPSwe] and human presenilin 1 [PS1]) harboring CCR2-deficient BMCs. Indeed, transplantation of CCR2-deficient BMCs enhanced the mnesic deficit and increased the amount of soluble Aβ and expression of transforming growth factor (TGF)-β1 and TGF-β receptors. By contrast, transplantation of wild-type bone marrow stem cells restored memory capacities and diminished soluble Aβ accumulation in APP_Swe/PS1 and APP_Swe/PS1/CCR2^−/− mice. Finally, gene therapy using a lentivirus-expressing CCR2 transgene in BMCs prevented cognitive decline in this mouse model of AD. Injection of CCR2 lentiviruses restored CCR2 expression and functions in monocytes. The presence of these cells in the brain of non-irradiated APP_Swe/PS1/CCR2^−/− mice supports the concept that they can be used as gene vehicles for AD. Decreased CCR2 expression in bone marrow–derived microglia may therefore play a major role in the etiology of this neurodegenerative disease.     

D5 dopamine receptor knockout mice and hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D1, D2, D3, D4, and D5) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D1-like receptors (D1 and D5) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D1-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D1-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D1 receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D5 receptor is also important in blood pressure regulation. Disruption of the D5 or the D1 receptor gene in mice increases blood pressure. However, unlike the D1 receptor, the hypertension in D5 receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V1 vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.