Pathological angiogenesis is a key feature of many diseases including retinopathies such as ROP (retinopathy of prematurity) and DR (diabetic retinopathy). There is considerable evidence that ...increased production of ROS (reactive oxygen species) in the retina participates in retinal angiogenesis, although the mechanisms by which this occurs are not fully understood. ROS is produced by a number of pathways, including the mitochondrial electron transport chain, cytochrome P450, xanthine oxidase and uncoupled nitric oxide synthase. The family of NADPH oxidase (Nox) enzymes are likely to be important given that their primary function is to produce ROS. Seven isoforms of Nox have been identified named Nox1-5, Duox (dual oxidase) 1 and Duox2. Nox1, Nox2 and Nox4 have been most extensively studied and are implicated in the development of conditions such as hypertension, cardiovascular disease and diabetic nephropathy. In recent years, evidence has accumulated to suggest that Nox1, Nox2 and Nox4 participate in pathological angiogenesis; however, there is no clear consensus about which Nox isoform is primarily responsible. In terms of retinopathy, there is growing evidence that Nox contribute to vascular injury. The RAAS (renin-angiotensin-aldosterone system), and particularly AngII (angiotensin II), is a key stimulator of Nox. It is known that a local RAAS exists in the retina and that blockade of AngII and aldosterone attenuate pathological angiogenesis in the retina. Whether the RAAS influences the production of ROS derived from Nox in retinopathy is yet to be fully determined. These topics will be reviewed with a particular emphasis on ROP and DR.
The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited ...by the lack of animal models that reproduce plaque instability observed in humans.
Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis.
On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E-deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques.
The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.
Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of ...its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE(-/-)) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 10(6) or 5 × 10(7) conventional B2 B cells but not 5 × 10(6) B1 B cells to a lymphocyte-deficient ApoE(-/-) Rag-2(-/-) common cytokine receptor γ-chain-deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 10(6) B2 B cells to an ApoE(-/-) mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.
Neovascular retinopathies are major causes of vision loss; yet treatments to prevent the condition are inadequate. The role of regulatory T cells in neovascular retinopathy is unknown. Here we show ...that in retinopathy regulatory T cells are transiently increased in lymphoid organs and the retina, but decline when neovascularization is established. The decline is prevented following regulatory T cells expansion with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of regulatory T cells. Further, both approaches reduce vasculopathy (vaso-obliteration, neovascularization, vascular leakage) and alter the activation of Tmem119
retinal microglia. Our in vitro studies complement these findings, showing that retinal microglia co-cultured with regulatory T cells exhibit a reduction in co-stimulatory molecules and pro-inflammatory mediators that is attenuated by CTLA-4 blockade. Collectively, we demonstrate that regulatory T cells are recruited to the retina and, when expanded in number, repair the vasculature. Manipulation of regulatory T cell numbers is a previously unrecognized, and promising avenue for therapies to prevent blinding neovascular retinopathies.The local immune responses in the eye are attenuated to preserve sight. Surprisingly, Deliyanti et al. show that regulatory T cells (Tregs) take an active role in protecting the eye from neovascularization in oxygen-induced retinopathy, and that interventions that augment the retinal Treg numbers reduce neovascular retinopathy in mice.
High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We ...investigated its role in the development of atherosclerosis in ApoE-/- mice.
Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE-/- mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-α and interleukin-1β tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers.
HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.
Atherosclerosis is driven by inflammatory reactions that are shared with the innate immune system. Toll-like receptor-9 (TLR9) is an intracellular pattern recognition receptor of the innate immune ...system that is currently under clinical investigation as a therapeutic target in inflammatory diseases. Here, we investigated whether TLR9 has a role in the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice.
Newly generated double-knockout ApoE(-/-):TLR9(-/-) mice and control ApoE(-/-) mice were fed a high-fat diet from 8 weeks and effects on lesion size, cellular composition, inflammatory status, and plasma lipids were assessed after 8, 12, 15, and 20 weeks. All 4 time points demonstrated exacerbated atherosclerotic lesion severity in ApoE(-/-):TLR9(-/-) mice, with a corresponding increase in lipid deposition and accumulation of macrophages, dendritic cells, and CD4(+) T cells. Although ApoE(-/-):TLR9(-/-) mice exhibited an increase in plasma very low-density lipoprotein/low-density-lipoprotein cholesterol, the very low-density lipoprotein/low-density lipoprotein:high-density lipoprotein ratio was unaltered because of a parallel increase in plasma high-density lipoprotein cholesterol. As a potential mechanism accounting for plaque progression in ApoE(-/-):TLR9(-/-) mice, CD4(+) T-cell accumulation was further investigated and depletion of these cells in ApoE(-/-):TLR9(-/-) mice significantly reduced lesion severity. As a final translational approach, administration of a TLR9 agonist (type B CpG oligodeoxynucleotide 1668) to ApoE(-/-) mice resulted in a reduction of lesion severity.
Genetic deletion of the innate immune receptor TLR9 exacerbated atherosclerosis in ApoE(-/-) mice fed a high-fat diet. CD4(+) T cells were identified as potential mediators of this effect. A type B CpG oligodeoxynucleotide TLR9 agonist reduced lesion severity, thus identifying a novel therapeutic approach in atherosclerosis.
NKT cells promote atherogenesis, but the subtypes responsible have not been identified. We investigated 2 major NKT cell subtypes (CD4+ and DN NKT) in ApoE-/- mice rendered NKT cell-deficient by ...day-3 neonatal thymectomy (3dTx).
Atherosclerosis development was studied in thymectomized ApoE-/- mice fed a high-fat diet with/without adoptively transferred NKT cells. We demonstrate NKT cell deficiency in thymectomized mice and markedly smaller atherosclerotic lesions. The reduction in lesion size was reversed by adoptive transfer of liver-derived NKT cells. Adoptive transfer of CD4+, but not DN NKT cells, into 3dTx ApoE-/- mice increased lesion size 2.5-fold. The differential effects were not attributable to differences in homing to developing atherosclerotic lesions. DN NKT cells expressed at least 3-fold higher levels of inhibitory Ly49 receptors (Ly49A, Ly49C/I, and Ly49G2) than CD4+ NKT cells, and lesions expressed large amounts of their MHC class I ligand. In vitro these inhibitory receptors initiated greater effects in DN NKT cells. Culture of each NKT cell subset with TAP-deficient (MHC class I-deficient) dendritic cells and alpha-GalCer led to secretion of similar amounts of proatherogenic cytokines IL-2, IFN-gamma, and TNF but, when cultured with MHC class I-positive dendritic cells, CD4+ NKT cells secreted more of these cytokines.
CD4+ NKT cells are responsible for the proatherogenic activity of NKT cells. Expression of inhibitory Ly49 receptors by the subtypes appears responsible for regulating their secretion of proatherogenic cytokines and their differential proatherogenic effects.
The postural tachycardia syndrome (POTS) has multiple symptoms, chief among which are tachycardia, weakness, and recurrent blackouts while standing. Previous research has implicated dysfunction of ...the norepinephrine transporter. A coding mutation in the norepinephrine transporter gene (SLC6A2) sequence has been reported in 1 family kindred only. The goal of the present study was to further characterize the role and regulation of the SLC6A2 gene in POTS.
Sympathetic nervous system responses to head-up tilt were examined by combining norepinephrine plasma kinetics measurements and muscle sympathetic nerve activity recordings in patients with POTS compared with that in controls. The SLC6A2 gene sequence was investigated in leukocytes from POTS patients and healthy controls using single nucleotide polymorphisms genotyping, bisulphite sequencing, and chromatin immunoprecipitation assays for histone modifications and binding of the transcriptional regulatory complex, methyl-CpG binding protein 2. The expression of norepinephrine transporter was lower in POTS patients compared with healthy volunteers. In the absence of altered SLC6A2 gene sequence or promoter methylation, this reduced expression was directly correlated with chromatin modifications.
We propose that chromatin-modifying events associated with SLC6A2 gene suppression may constitute a mechanism of POTS.
Summary
Angiogenesis and inflammation are causative factors in the development of neovascular retinopathies. These processes involve the retinal endothelium and the retinal immune cells, microglia. ...The renin‐angiotensin system contributes to retinal injury via the actions of the type 1 angiotensin receptor (AT1R). However, it has been suggested that prorenin, the initiator of the renin‐angiotensin system cascade, influences retinal injury independently from the AT1R. We evaluated whether prorenin induced a pro‐angiogenic and pro‐inflammatory response in retinal endothelial cells and a pro‐inflammatory phenotype in retinal microglia. Primary cultures of retinal endothelial cells and microglia were studied. Rat recombinant prorenin (2 nmol/L) stimulated the proliferation and tubulogenesis of retinal endothelial cells; it increased the levels of pro‐angiogenic factors, vascular endothelial growth factor, angiopoietin‐1, and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains, and pro‐inflammatory factors, intercellular adhesion molecule‐1 and monocyte chemoattractant protein‐1, relative to the controls. The messenger RNA levels of the (pro)renin receptor were also increased. These effects occurred in the presence of the AT1R blocker candesartan (10 μmol/L) and the renin inhibitor aliskiren (10 μmol/L). Microglia, which express the (pro)renin receptor, elicited an activated phenotype when exposed to prorenin, which was characterized by increased levels of intercellular adhesion molecule‐1, monocyte chemoattractant protein‐1, tumour necrosis factor‐α, interleukin‐6, and interleukin‐1β and by decreased levels of interleukin‐10 and arginase‐1 relative to controls. Candesartan did not influence the effects of prorenin on retinal microglia. In conclusion, prorenin has distinct pro‐angiogenic and pro‐inflammatory effects on retinal cells that are independent of the AT1R, indicating the potential importance of prorenin in retinopathy.
To determine the significance of fibroblast growth factor receptor (FGFR) expression for the development of atherosclerotic lesions in apoE-deficient (apoE-/-) mice.
ApoE-/- mice fed a high-fat diet ...were administered the FGFR tyrosine kinase inhibitor SU5402 (25 mg/kg/d sc), which inhibited neointima growth by 85%. We measured its effects on lesion size at the aortic sinus, macrophage and smooth muscle cell (SMC) accumulation, the expression of monocyte chemotactic and retention factors, as well as its effects on FGFR expression/phosphorylation. FGFR tyrosine kinase inhibition reduced phosphorylated FGFRs in lesions by 90%, associated with a 65% reduction in lesion size measured using Oil Red O. Macrophages and SMCs within lesions were reduced by 58% and 78%, respectively. Monocyte chemotactic protein-1 (MCP-1) expression was also reduced, as was the expression of hyaluronan synthase, cyclooxygenase-2, CD36, and endothelial monocyte-activating polypeptide-II. Although 3 FGFR types were expressed in lesions, the effects of SU5402 could be attributed largely to inhibition of FGFR-1 phosphorylation.
Atherosclerotic lesions in apoE-/- mice express multiple FGFRs and an active FGF:FGFR-1 signaling system that promotes atherosclerosis development via increased SMC proliferation, and by augmenting macrophage accumulation via increased expression of MCP-1 and factors promoting macrophage retention in lesions.
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