Eileen McNeill

Galectin-3 Is an Amplifier of Inflammation in Atherosclerotic Plaque Progression Through Macrophage Activation And Monocyte Chemoattraction

Objective—Galectin-3 (Gal-3) is a 26-kDa lectin known to regulate many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to atherosclerotic plaque progression by enhancing monocyte chemoattraction through macrophage activation. Methods and Results—Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE−/− mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3–treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), an effect that was reduced by 66 and 60% by Pertussis Toxin (PTX) and the Vaccinia virus protein 35K, respectively. Microarray analysis of human macrophages and subsequent qPCR validation confirmed the upregulation of CC chemokines in response to Gal-3 treatment. Conclusions—Our data suggest that Gal-3 is both a marker of atherosclerotic plaque progression and a central contributor to the pathology by amplification of key proinflammatory molecules.

Anti-inflammatory effects of nicotinic acid in adipocytes demonstrated by suppression of fractalkine, RANTES, and MCP-1 and upregulation of adiponectin

Objective A major site of action for the atheroprotective drug nicotinic acid (NA) is adipose tissue, via the G-protein-coupled receptor, GPR109A. Since, adipose tissue is an active secretory organ that contributes both positively and negatively to systemic inflammatory processes associated with cardiovascular disease, we hypothesized that NA would act directly upon adipocytes to alter the expression of pro-inflammatory chemokines, and the anti-inflammatory adipokine adiponectin. Methods and results TNF-α treatment (1.0 ng/mL) of 3T3-L1 adipocytes resulted in an increase in gene expression of fractalkine (9 ± 3.3-fold, P < 0.01); monocyte chemoattractant protein-1 (MCP-1) (24 ± 1.2-fold, P < 0.001), ‘regulated upon activation, normal T cell expressed and secreted’ (RANTES) (500 ± 55-fold, P < 0.001) and inducible nitric oxide synthase (iNOS) (200 ± 70-fold, P < 0.05). The addition of NA (10−4 M) to TNF-α-treated adipocytes attenuated expression of fractalkine (50 ± 12%, P < 0.01); MCP-1 (50 ± 6%, P < 0.01), RANTES (70 ± 3%, P < 0.01) and iNOS (60 ± 16%). This pattern was mirrored in protein released from the adipocytes into the surrounding media. The effect on gene expression was neutralised by pre-treatment with pertussis toxin. NA attenuated macrophage chemotaxis (by 27 ± 3.5%, P < 0.001) towards adipocyte conditioned media. By contrast, NA, (10−6–10−3 M) increased, in a dose-dependent manner, mRNA of the atheroprotective hormone adiponectin (3–5-fold n = 6, P < 0.01). Conclusions NA suppresses pro-atherogenic chemokines and upregulates the atheroprotective adiponectin through a G-protein-coupled pathway. Since adipose tissue has the potential to contribute to both systemic and local (perivascular) inflammation associated with atherosclerosis our results suggest a new “pleiotropic” role for NA.

Tetrahydrobiopterin in Cardiovascular Health and Disease

Tetrahydrobiopterin (BH4) functions as a cofactor for several important enzyme systems, and considerable evidence implicates BH4 as a key regulator of endothelial nitric oxide synthase (eNOS) in the setting of cardiovascular health and disease. BH4 bioavailability is determined by a balance of enzymatic de novo synthesis and recycling, versus degradation in the setting of oxidative stress. Augmenting vascular BH4 levels by pharmacological supplementation has been shown in experimental studies to enhance NO bioavailability. However, it has become more apparent that the role of BH4 in other enzymatic pathways, including other NOS isoforms and the aromatic amino acid hydroxylases, may have a bearing on important aspects of vascular homeostasis, inflammation, and cardiac function. This article reviews the role of BH4 in cardiovascular development and homeostasis, as well as in pathophysiological processes such as endothelial and vascular dysfunction, atherosclerosis, inflammation, and cardiac hypertrophy. We discuss the therapeutic potential of BH4 in cardiovascular disease states and attempt to address how this modulator of intracellular NO-redox balance may ultimately provide a powerful new treatment for many cardiovascular diseases.

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

Aims Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Methods and results Endothelial cell repopulation was assessed en face in stented arteries in ApoE−/− mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm2, P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm2, P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE−/− mice had less neointima formation compared with ApoE−/− littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm2, P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm2, P = 0.043). Conclusion Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Inflammatory cell recruitment in cardiovascular disease: murine models and potential clinical applications

Atherosclerosis is the pathological process that underlies the development of cardiovascular disease, a leading cause of mortality. Atherosclerotic plaque formation is driven by the recruitment of inflammatory monocytes into the artery wall, their differentiation into macrophages and the subsequent transformation of macrophages into cholesterol-laden foam cells. Models of hypercholesterolaemia such as the ApoE (apolipoprotein E)-/- mouse and the application of transgenic technologies have allowed us to undertake a thorough dissection of the cellular and molecular biology of the atherosclerotic disease process. Murine models have emphasized the central role of inflammation in atherogenesis and have been instrumental in the identification of adhesion molecules that support monocyte recruitment, scavenger receptors that facilitate cholesterol uptake by macrophages and other macrophage activation receptors. The study of mice deficient in multiple members of the chemokine family, and their receptors, has shown that chemokines play a critical role in promoting atherosclerotic plaque formation. In the present review, we will discuss novel therapeutic avenues for the treatment of cardiovascular disease that derive directly from our current understanding of atherogenesis gained in experimental animal models.

Endothelial Cell-Specific ROS Production Increases Susceptibility to Aortic Dissection

Background— Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results— A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions— These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

Endothelial Cell–Specific Reactive Oxygen Species Production Increases Susceptibility to Aortic Dissection

Background— Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results— A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions— These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation.

Cell-Autonomous Role of Endothelial GTP Cyclohydrolase 1 and Tetrahydrobiopterin in Blood Pressure Regulation

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell–specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1fl/fl) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1fl/flTie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2•– production. GCH1fl/flTie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1fl/flTie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1fl/flTie2cre aortas. GCH1fl/flTie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.