Anita C. Thomas

Nitric oxide synthase: non-canonical expression patterns

Science can move ahead by questioning established or canonical views and, so it may be with the enzymes, nitric oxide synthases (NOS). Nitric oxide (NO) is generated by NOS isoforms that are often described by their tissue-specific expression patterns. NOS1 (nNOS) is abundant in neural tissue, NOS2 is upregulated in activated macrophages and known as inducible NOS (iNOS), and NOS3 (eNOS) is abundant in endothelium where it regulates vascular tone. These isoforms are described as constitutive or inducible, but in this perspective we question the broad application of these labels. Are there instances where “constitutive” NOS (NOS1 and NOS3) are inducibly expressed; conversely, are there instances where NOS2 is constitutively expressed? NOS1 and NOS3 inducibility may be linked to post-translational regulation, making their actual patterns activity much more difficult to detect. Constitutive NOS2 expression has been observed in several tissues, especially the human pulmonary epithelium where it may regulate airway tone. These data suggest that expression of the three NOS enzymes may include non-established patterns. Such information should be useful in designing strategies to modulate these important enzymes in different disease states.

Macrophage: SHIP of Immunity

Immunology. Why does it exist? Two words. Cure disease. People get diseases. “Test tubes” do not. People fund immunologists for solutions to their health problems. But, immunologists often study leukocytes in test tubes – the laboratory – away from diseases. Why? Because much can be learned from analyzing cellular biochemistry and behaviors in vitro that cannot be ascertained when leukocytes are in animals. At the same time, isolated leukocyte reactions often do not reflect how the immune system operates as a unit. So, it is critical to verify in vitro observations in vivo. Among leukocytes, macrophages are the central initiating and directing element in immune systems, and serve this role through four basic “SHIP” functions in vivo: Sample; Heal; Inhibit; and Present (antigen) (1–4). The polar-opposite functions of Heal (M2-type) and Inhibit (M1-type) can have profoundly different effects on host survival, and require unique and major changes in macrophage metabolism and physiology. In turn, macrophage populations are necessarily heterogeneous as they adapt to protect hosts in different ways: they exhibit “plasticity.” Some have focused on measuring ever-expanding lists of cell surface or various other “markers” (mostly in vitro) to try and sub-type macrophages. But, the “heterogeneity” created by such studies can be “illusory” because there are many more markers than there are functions (e.g., M1/inhibit and M2/heal). Thus, it is important to focus on classifying macrophages by functions, such as SHIP, to navigate through a “sea of plasticity.” And, thereby realize the enormous potential of macrophages/innate immunity for improving health.

Classical and Alternative Activation and Metalloproteinase Expression Occurs in Foam Cell Macrophages in Male and Female ApoE Null Mice in the Absence of T and B Lymphocytes.

Background: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. Methods and results: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice. Conclusion: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.

Cytochrome P4502S1: a novel monocyte/macrophage fatty acid epoxygenase in human atherosclerotic plaques

Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E2 is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG2 and PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE−/− mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.

Animal models for studying vein graft failure and therapeutic interventions.

Vein grafts have been extensively used to bypass blockages in arteries, but are themselves subject to early closure by thrombosis or later obstruction by vein graft disease (neointimal hyperplasia and remodelling). Animal models are a crucial means of testing potential therapeutic and surgical interventions to prevent graft stenosis and occlusion. This review outlines many of the animal models of vein grafting. Recent studies include targeted gene therapy to prevent acute vein graft thrombosis and the use of folic acid to limit graft failure in diabetic pigs.

Plaque Size Is Decreased but M1 Macrophage Polarization and Rupture Related Metalloproteinase Expression Are Maintained after Deleting T-Bet in ApoE Null Mice.

Background Thelper1 (Th1) lymphocytes have been previously implicated in atherosclerotic plaque growth but their role in plaque vulnerability to rupture is less clear. We investigated whether T-bet knockout that prevents Th1 lymphocyte differentiation modulates classical (M1) macrophage activation or production of matrix degrading metalloproteinases (MMPs) and their tissue inhibitors, TIMPs. Methods & Results We studied the effect of T-bet deletion in apolipoproteinE (ApoE) knockout mice fed a high fat diet (HFD) or normal chow diet (ND). Transcript levels of M1/M2 macrophage polarization markers, selected MMPs and TIMPs were measured by RT-qPCR in macrophages isolated from subcutaneous granulomas or in whole aortae. Immunohistochemistry of aortic sinus (AS) and brachiocephalic artery (BCA) plaques was conducted to quantify protein expression of the same factors. Deletion of T-bet decreased mRNA for the M1 marker NOS-2 in granuloma macrophages but levels of M2 markers (CD206, arginase-1 and Ym-1), MMPs-2, -9, -12, -13, -14 and -19 or TIMPs-1 to -3 were unchanged. No mRNA differences were observed in aortic extracts from mice fed a HFD for 12 weeks. Moreover, AS and BCA plaques were similarly sized between genotypes, and had similar areas stained for NOS-2, COX-2, MMP-12 and MMP-14 proteins. T-bet deletion increased MMP-13, MMP-14 and arginase-1 in AS plaques. After 35 weeks of ND, T-bet deletion reduced the size of AS and BCA plaques but there were no differences in the percentage areas stained for M1 or M2 markers, MMPs-12, -13, -14, or TIMP-3. Conclusions Absence of Th1 lymphocytes is associated with reduced plaque size in ApoE knockout mice fed a normal but not high fat diet. In either case, M1 macrophage polarization and expression of several MMPs related to plaque instability are either maintained or increased.

Layered double hydroxide nanoparticles: Impact on vascular cells, blood cells and the complement system

The mounting interest in layered double hydroxide (LDH) nanoparticles as drug carriers and bio-imaging contrast agents makes biosafety evaluation of LDH essential. Considering the important role of blood circulation in bio-distribution of nanoparticles, the present work evaluated the impact of MgAl-LDHs on key components of the circulatory system, including vascular cells (vascular smooth muscle cells (SMCs) and endothelial cells (HUVECs)), red blood cells (RBCs), and complement activation. The results showed that LDH had no effects on SMCs and HUVECs at concentrations up to 500 and 10u202fµg/mL respectively, in terms of cell proliferation and viability. LDH (10u202fµg/mL) did not change either the migration distance or the number of migrating SMCs in culture. Moreover, LDH (400u202fµg/mL) had a negligible effect on RBCs lysis, and there was no significant increase in levels of complement activation product, C5a, in the presence of LDH (20 or 200u202fµg/mL). The low toxicity for vascular cells and blood cells combined with low immunogenicity sheds a light on the biosafety of LDH nanoparticles, and encourages further studies into their biomedical applications.

The pro-fibrotic and anti-inflammatory foam cell macrophage paradox

The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity.

A humanized HLA-DR4 mouse model for autoimmune myocarditis

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3 weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment.

Strategies to Extend the Life of Saphenous Vein Grafts

Saphenous veins are widely used to bypass blockages in coronary and peripheral arteries, but up to 50 % of grafts fail within 10–15 years. This is due to neointimal formation and vein graft disease, processes involving the proliferation of vascular smooth muscle cells, the production of extracellular matrix, and superimposed atherosclerosis. Several approaches aimed at preventing neointimal formation and extending the life of vein grafts have been devised that have yielded promising results. In this chapter we will briefly summarise the pathophysiology of vein graft disease and then consider potential interventions to prevent vein graft failure, such as changes in lifestyle, surgical technique, pharmacological approaches, external sheaths, and gene or stem cell transfer.