Anti-Galectin-2 Antibody Treatment Reduces Atherosclerotic Plaque Size and Alters Macrophage Polarity.

BACKGROUND: Galectins have numerous cellular functions in immunity and inflammation. Short-term galectin-2 (Gal-2) blockade in ischemia-induced arteriogenesis shifts macrophages to an anti-inflammatory phenotype and improves perfusion. Gal-2 may also affect other macrophage-related cardiovascular diseases. OBJECTIVES: This study aims to elucidate the effects of Gal-2 inhibition in atherosclerosis. METHODS: ApoE -/- mice were given a high-cholesterol diet (HCD) for 12 weeks. After 6 weeks of HCD, intermediate atherosclerotic plaques were present. To study the effects of anti-Gal-2 nanobody treatment on the progression of existing atherosclerosis, treatment with two llama-derived anti-Gal-2 nanobodies (clones 2H8 and 2C10), or vehicle was given for the remaining 6 weeks. RESULTS: Gal-2 inhibition reduced the progression of existing atherosclerosis. Atherosclerotic plaque area in the aortic root was decreased, especially so in mice treated with 2C10 nanobodies. This clone showed reduced atherosclerosis severity as reflected by a decrease in fibrous cap atheromas in addition to decreases in plaque size.The number of plaque resident macrophages was unchanged; however, there was a significant increase in the fraction of CD206+ macrophages. 2C10 treatment also increased plaque α-smooth muscle content, and Gal-2 may have a role in modulating the inflammatory status of smooth muscle cells. Remarkably, both treatments reduced serum cholesterol concentrations including reductions in very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein while triglyceride concentrations were unchanged. CONCLUSION: Prolonged and frequent treatment with anti-Gal-2 nanobodies reduced plaque size, slowed plaque progression, and modified the phenotype of plaque macrophages toward an anti-inflammatory profile. These results hold promise for future macrophage modulating therapeutic interventions that promote arteriogenesis and reduce atherosclerosis.

Coronary Collateral Flow Index Is Correlated With the Palmar Collateral Flow Index: Indicating Systemic Collateral Coherence in Individual Patients-Brief Report.

[Figure: see text].

Stimulation of Collateral Vessel Growth by Inhibition of Galectin 2 in Mice Using a Single-Domain Llama-Derived Antibody.

Background In the presence of arterial stenosis, collateral artery growth (arteriogenesis) can alleviate ischemia and preserve tissue function. In patients with poorly developed collateral arteries, Gal-2 (galectin 2) expression is increased. In vivo administration of Gal-2 inhibits arteriogenesis. Blocking of Gal-2 potentially stimulates arteriogenesis. This study aims to investigate the effect of Gal-2 inhibition on arteriogenesis and macrophage polarization using specific single-domain antibodies. Methods and Results Llamas were immunized with Gal-2 to develop anti-Gal-2 antibodies. Binding of Gal-2 to monocytes and binding inhibition of antibodies were quantified. To test arteriogenesis in vivo, Western diet-fed LDLR.(low-density lipoprotein receptor)-null Leiden mice underwent femoral artery ligation and received treatment with llama antibodies 2H8 or 2C10 or with vehicle. Perfusion restoration was measured with laser Doppler imaging. In the hind limb, arterioles and macrophage subtypes were characterized by histology, together with aortic atherosclerosis. Llama-derived antibodies 2H8 and 2C10 strongly inhibited the binding of Gal-2 to monocytes (93% and 99%, respectively). Treatment with these antibodies significantly increased perfusion restoration at 14 days (relative to sham, vehicle: 41.3±2.7%; 2H8: 53.1±3.4%, P=0.016; 2C10: 52.0±3.8%, P=0.049). In mice treated with 2H8 or 2C10, the mean arteriolar diameter was larger compared with control (vehicle: 17.25±4.97 µm; 2H8: 17.71±5.01 µm; 2C10: 17.84±4.98 µm; P<0.001). Perivascular macrophages showed a higher fraction of the M2 phenotype in both antibody-treated animals (vehicle: 0.49±0.24; 2H8: 0.73±0.15, P=0.007; 2C10: 0.75±0.18, P=0.006). In vitro antibody treatment decreased the expression of M1-associated cytokines compared with control (P<0.05 for each). Atherosclerotic lesion size was comparable between groups (overall P=0.59). Conclusions Inhibition of Gal-2 induces a proarteriogenic M2 phenotype in macrophages, improves collateral artery growth, and increases perfusion restoration in a murine hind limb model.

Elevated monocyte-specific type I interferon signalling correlates positively with cardiac healing in myocardial infarct patients but interferon alpha application deteriorates myocardial healing in rats.

Monocytes are involved in adverse left ventricular (LV) remodelling following myocardial infarction (MI). To provide therapeutic opportunities we aimed to identify gene transcripts in monocytes that relate to post-MI healing and evaluated intervention with the observed gene activity in a rat MI model. In 51 MI patients treated by primary percutaneous coronary intervention (PCI), the change in LV end-diastolic volume index (EDVi) from baseline to 4-month follow-up was assessed using cardiovascular magnetic resonance imaging (CMR). Circulating monocytes were collected at day 5 (Arterioscler Thromb Vasc Biol 35:1066-1070, 2015; Cell Stem Cell 16:477-487, 2015; Curr Med Chem 13:1877-1893, 2006) after primary PCI for transcriptome analysis. Transcriptional profiling and pathway analysis revealed that patients with a decreased LV EDVi showed an induction of type I interferon (IFN) signalling (type I IFN pathway: P value < 0.001; false discovery rate < 0.001). We subsequently administered 15,000 Units of IFN-α subcutaneously in a rat MI model for three consecutive days following MI. Cardiac function was measured using echocardiography and infarct size/cardiac inflammation using (immuno)-histochemical analysis. We found that IFN-α application deteriorated ventricular dilatation and increased infarct size at day 28 post-MI. Moreover, IFN-α changed the peripheral monocyte subset distribution towards the pro-inflammatory monocyte subset whereas in the myocardium, the presence of the alternative macrophage subset was increased at day 3 post-MI. Our findings suggest that induction of type I IFN signalling in human monocytes coincides with adverse LV remodelling. In rats, however, IFN-α administration deteriorated post-MI healing. These findings underscore important but also contradictory roles for the type I IFN response during cardiac healing following MI.

Correction: Dissecting the Effects of Ischemia and Reperfusion on the Coronary Microcirculation in a Rat Model of Acute Myocardial Infarction.

[This corrects the article DOI: 10.1371/journal.pone.0157233.].

Dissecting the Effects of Ischemia and Reperfusion on the Coronary Microcirculation in a Rat Model of Acute Myocardial Infarction.

BACKGROUND: Microvascular injury (MVI) after coronary ischemia-reperfusion is associated with high morbidity and mortality. Both ischemia and reperfusion are involved in MVI, but to what degree these phases contribute is unknown. Understanding the etiology is essential for the development of new potential therapies. METHODS AND FINDINGS: Rats were divided into 3 groups receiving either 30 minutes ischemia, 90 minutes ischemia or 30 minutes ischemia followed by 60 minutes reperfusion. Subsequently hearts were ex-vivo perfused in a Langendorff-model. Fluorescence and electron microscopy was used for analysis of capillary density, vascular permeability and ultrastructure. Most MVI was observed after 30 minutes ischemia followed by 60 minutes reperfusion. In comparison to the 30' and 90' ischemia group, wall thickness decreased (207.0±74 vs 407.8±75 and 407.5±71, p = 0.02). Endothelial nuclei in the 30'-60' group showed irreversible damage and decreased chromatin density variation (50.5±9.4, 35.4±7.1 and 23.7±3.8, p = 0.03). Cell junction density was lowest in the 30'-60' group (0.15±0.02 vs 2.5±0.6 and 1.8±0.7, p<0.01). Microsphere extravasation was increased in both the 90' ischemia and 30'-60' group. CONCLUSIONS: Ischemia alone for 90 minutes induces mild morphological changes to the coronary microcirculation, with increased vascular permeability. Ischemia for 30 minutes, followed by 60 minutes of reperfusion, induces massive MVI. This shows the direct consequences of reperfusion on the coronary microcirculation. These data imply that a therapeutic window exists to protect the microcirculation directly upon coronary revascularization.

CD40 in coronary artery disease: a matter of macrophages?

Coronary artery disease (CAD), also known as ischemic heart disease (IHD), is the leading cause of mortality in the western world, with developing countries showing a similar trend. With the increased understanding of the role of the immune system and inflammation in coronary artery disease, it was shown that macrophages play a major role in this disease. Costimulatory molecules are important regulators of inflammation, and especially, the CD40L-CD40 axis is of importance in the pathogenesis of cardiovascular disease. Although it was shown that CD40 can mediate macrophage function, its exact role in macrophage biology has not gained much attention in cardiovascular disease. Therefore, the goal of this review is to give an overview on the role of macrophage-specific CD40 in cardiovascular disease, with a focus on coronary artery disease. We will discuss the function of CD40 on the macrophage and its (proposed) role in the reduction of atherosclerosis, the reduction of neointima formation, and the stimulation of arteriogenesis.

The emerging role of galectins in cardiovascular disease.

Galectins are an ancient family of ß-galactoside-specific lectins and consist of 15 different types, each with a specific function. They play a role in the immune system, inflammation, wound healing and carcinogenesis. In particular the role of galectin in cancer is widely studied. Lately, the role of galectins in the development of cardiovascular disease has gained attention. Worldwide cardiovascular disease is still the leading cause of death. In ischemic heart disease, atherosclerosis limits adequate blood flow. Angiogenesis and arteriogenesis are highly important mechanisms relieving ischemia by restoring perfusion to the post-stenotic myocardial area. Galectins act ambiguous, both relieving ischemia and accelerating atherosclerosis. Atherosclerosis can ultimately lead to myocardial infarction or ischemic stroke, which are both associated with galectins. There is also a role for galectins in the development of myocarditis by their influence on inflammatory processes. Moreover, galectin acts as a biomarker for the severity of myocardial ischemia and heart failure. This review summarizes the association between galectins and the development of multiple cardiovascular diseases such as myocarditis, ischemic stroke, myocardial infarction, heart failure and atrial fibrillation. Furthermore it focuses on the association between galectin and more general mechanisms such as angiogenesis, arteriogenesis and atherosclerosis.

Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage.

RATIONALE: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. OBJECTIVES: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. METHODS: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. MEASUREMENTS AND MAIN RESULTS: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. CONCLUSIONS: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

IFN-ß affects the angiogenic potential of circulating angiogenic cells by activating calpain 1.

Circulating angiogenic cells (CACs) are monocyte-derived cells with endothelial characteristics, which contribute to both angiogenesis and arteriogenesis in a paracrine way. Interferon-ß (IFN-ß) is known to inhibit these divergent processes in animals and patients. We hypothesized that IFN-ß might act by affecting the differentiation and function of CACs. CACs were cultured from peripheral blood mononuclear cells and phenotypically characterized by surface expression of monocytic and endothelial markers. IFN-ß significantly reduced the number of CACs by 18-64%. Apoptosis was not induced by IFN-ß, neither in mononuclear cells during differentiation, nor after maturation to CACs. Rather, IFN-ß impaired adhesion to, and spreading on, fibronectin, which was dependent on α5ß1 (VLA-5)-integrin. IFN-ß affected the function of VLA-5 in mature CACs, leading to rounding and detachment of cells, by induction of calpain 1 activity. Cell rounding and detachment was completely reversed by inhibition of calpain 1 activity in mature CACs. During in vitro capillary formation, CAC addition and calpain 1 inhibition enhanced sprouting of endothelial cells to a comparable extent, but were not sufficient to rescue tube formation in the presence of IFN-ß. We show that the IFN-ß-induced reduction of the numbers of in vitro differentiated CACs is based on activation of calpain 1, resulting in an attenuated adhesion to extracellular matrix proteins via VLA-5. In vivo, this could lead to inhibition of vessel formation due to reduction of the locally recruited CAC numbers and their paracrine angiogenic factors.

Palmitic acid increases pro-oxidant adaptor protein p66Shc expression and affects vascularization factors in angiogenic mononuclear cells: Action of resveratrol.

A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control.

Metabolic changes in type 2 diabetes are reflected in peripheral blood cells, revealing aberrant cytotoxicity, a viral signature, and hypoxia inducible factor activity.

BACKGROUND: Metabolic syndrome (MetS) is characterized by central obesity, insulin resistance, dysglycemia, and a pro-atherogenic plasma lipid profile. MetS creates a high risk for development of type 2 diabetes (T2DM) and cardiovascular disease (CVD), presumably by altering inflammatory responses. Presently, it is unknown how the chronic metabolic disturbances in acute hyperglycemia, MetS and T2DM affect the immune activity of peripheral blood cells. METHODS: We performed genome-wide expression analysis of peripheral blood cells obtained from patients with T2DM (n = 6) and age-, sex- , BMI- and blood pressure-matched obese individuals with MetS (n = 4) and lean healthy normoglycemic controls (n = 3), both under fasting conditions and after controlled induction of acute hyperglycemia during a 70 min hyperglycemic clamp. Differential gene expression during fasting conditions was confirmed by real-time PCR, for which we included additional age-, sex-, BMI-, and blood pressure-matched obese individuals with (n = 4) or without (n = 4) MetS. RESULTS: Pathway and Gene ontology analysis applied to baseline expression profiles of peripheral blood cells from MetS and T2DM patients revealed metabolic changes, highly similar to a reoviral infection gene signature in T2DM patients. Transcription factor binding site analysis indicated that increased HIF-1α activity, a transcription factor induced by either hypoxia or oxidative stress, is responsible for this aberrant metabolic profile in peripheral blood cells from T2DM patients. Acute hyperglycemia in healthy controls resulted in reduced expression of cytotoxicity-related genes, representing NK- and CD8(+) cells. In obese controls, MetS and especially T2DM patients, baseline expression of genes involved in cytotoxicity was already low, compared to healthy controls and did not further decrease upon acute hyperglycemia. CONCLUSIONS: The reduced activity of cytotoxic genes in T2DM is explained by chronic hyperglycemia, but its acute effects are restricted to healthy controls. Genome expression of circulating leukocytes from T2DM patients differs from MetS individuals by a specific reovirus signature. Our data thus suggest a role for suppressed anti-viral capacity in the etiology of diabetes.

MAb therapy against the IFN-α/ß receptor subunit 1 stimulates arteriogenesis in a murine hindlimb ischaemia model without enhancing atherosclerotic burden.

AIMS: IFN-beta (IFNß) signalling is increased in patients with insufficient coronary collateral growth (i.e. arteriogenesis) and IFNß hampers arteriogenesis in mice. A downside of most pro-arteriogenic agents investigated in the past has been their pro-atherosclerotic properties, rendering them unsuitable for therapeutic application. Interestingly, type I IFNs have also been identified as pro-atherosclerotic cytokines and IFNß treatment increases plaque formation and accumulation of macrophages. We therefore hypothesized that mAb therapy to inhibit IFNß signalling would stimulate arteriogenesis and simultaneously attenuate-rather than aggravate-atherosclerosis. METHODS AND RESULTS: In a murine hindlimb ischaemia model, atherosclerotic low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated during a 4-week period with blocking MAbs specific for mouse IFN-α/ß receptor subunit 1 (IFNAR1) or murine IgG isotype as a control. The arteriogenic response was quantified using laser Doppler perfusion imaging (LDPI) as well as immunohistochemistry. Effects on atherosclerosis were determined by quantification of plaque area and analysis of plaque composition. Downstream targets of IFNß were assessed by real-time PCR (RT-PCR) in the aortic arch. Hindlimb perfusion restoration after femoral artery ligation was improved in mice treated with anti-IFNAR1 compared with controls as assessed by LDPI. This was accompanied by a decrease in CXCL10 expression in the IFNAR1 MAb-treated group. Anti-IFNAR1 treatment reduced plaque apoptosis without affecting total plaque area or other general plaque composition parameters. Results were confirmed in a short-term model and in apolipoprotein E knockout (APOE)(-/-) mice. CONCLUSION: Monoclonal anti-IFNAR1 therapy during a 4-week treatment period stimulates collateral artery growth in mice and did not enhance atherosclerotic burden. This is the first reported successful strategy using MAbs to stimulate arteriogenesis.

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Doppler-derived intracoronary physiology indices predict the occurrence of microvascular injury and microvascular perfusion deficits after angiographically successful primary percutaneous coronary intervention.

BACKGROUND: A total of 40% to 50% of patients with ST-segment-elevation myocardial infarction develop microvascular injury (MVI) despite angiographically successful primary percutaneous coronary intervention (PCI). We investigated whether hyperemic microvascular resistance (HMR) immediately after angiographically successful PCI predicts MVI at cardiovascular magnetic resonance and reduced myocardial blood flow at positron emission tomography (PET). METHODS AND RESULTS: Sixty patients with ST-segment-elevation myocardial infarction were included in this prospective study. Immediately after successful PCI, intracoronary pressure-flow measurements were performed and analyzed off-line to calculate HMR and indices derived from the pressure-velocity loops, including pressure at zero flow. Cardiovascular magnetic resonance and H2 (15)O PET imaging were performed 4 to 6 days after PCI. Using cardiovascular magnetic resonance, MVI was defined as a subendocardial recess of myocardium with low signal intensity within a gadolinium-enhanced area. Myocardial perfusion was quantified using H2 (15)O PET. Reference HMR values were obtained in 16 stable patients undergoing coronary angiography. Complete data sets were available in 48 patients of which 24 developed MVI. Adequate pressure-velocity loops were obtained in 29 patients. HMR in the culprit artery in patients with MVI was significantly higher than in patients without MVI (MVI, 3.33±1.50 mm Hg/cm per second versus no MVI, 2.41±1.26 mm Hg/cm per second; P=0.03). MVI was associated with higher pressure at zero flow (45.68±13.16 versus 32.01±14.98 mm Hg; P=0.015). Multivariable analysis showed HMR to independently predict MVI (P=0.04). The optimal cutoff value for HMR was 2.5 mm Hg/cm per second. High HMR was associated with decreased myocardial blood flow on PET (myocardial perfusion reserve <2.0, 3.18±1.42 mm Hg/cm per second versus myocardial perfusion reserve ≥2.0, 2.24±1.19 mm Hg/cm per second; P=0.04). CONCLUSIONS: Doppler-flow-derived physiological indices of coronary resistance (HMR) and extravascular compression (pressure at zero flow) obtained immediately after successful primary PCI predict MVI and decreased PET myocardial blood flow. CLINICAL TRIAL REGISTRATION URL: http://www.trialregister.nl. Unique identifier: NTR3164.

Interferon-Beta, a Decisive Factor in Angiogenesis and Arteriogenesis.

In this review we discuss the current literature on the effects of type I interferons (IFN) and their downstream effectors on vascular growth in experimental models in vitro and in vivo. In addition to its well-documented role in angiogenesis, that is, the growth of new capillaries from existing vessels, we will also describe emerging evidence and mechanisms by which type I IFN may inhibit arteriogenesis, that is, the expansive remodeling of existing collateral arteries. Crucial in both processes is the common role of circulating monocytes, which are known to act as pivotal cellular modulators in revascularization through secreted chemokines, proteases, and growth factors. These secreted molecules, which are all modulated by IFN signaling, act via degradation of the extracellular matrix and by stimulating the proliferation of vascular smooth muscle cells and endothelial cells. Thus, next to the antiviral and immunomodulatory activities of type I IFNs, a potent role of IFN-ß as modulator of revascularization is now emerging and may be considered a potential clinical target for the stimulation of angiogenesis and arteriogenesis in ill-perfused tissues.

PPARγ activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors.

BACKGROUND: Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs). METHODS: PACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 µmol/L) or GW9662 (10 µmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model. RESULTS: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells. CONCLUSIONS: In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

Systemic toll-like receptor and interleukin-18 pathway activation in patients with acute ST elevation myocardial infarction.

Acute myocardial infarction (AMI) is accompanied by increased expression of Toll like receptors (TLR)-2 and TLR4 on circulating monocytes. In animal models, blocking TLR2/4 signaling reduces inflammatory cell influx and infarct size. The clinical consequences of TLR activation during AMI in humans are unknown, including its role in long-term cardiac functional outcome Therefore, we analyzed gene expression in whole blood samples from 28 patients with an acute ST elevation myocardial infarction (STEMI), enrolled in the EXenatide trial for AMI patients (EXAMI), both at admission and after 4-month follow-up, by whole genome expression profiling and real-time PCR. Cardiac function was determined by cardiac magnetic resonance (CMR) imaging at baseline and after 4-month follow-up. TLR pathway activation was shown by increased expression of TLR4 and its downstream genes, including IL-18R1, IL-18R2, IL-8, MMP9, HIF1A, and NFKBIA. In contrast, expression of the classical TLR-induced genes, TNF, was reduced. Bioinformatics analysis and in vitro experiments explained this noncanonical TLR response by identification of a pivotal role for HIF-1α. The extent of TLR activation and IL-18R1/2 expression in circulating cells preceded massive troponin-T release and correlated with the CMR-measured ischemic area (R=0.48, p=0.01). In conclusion, we identified a novel HIF-1-dependent noncanonical TLR activation pathway in circulating leukocytes leading to enhanced IL-18R expression which correlated with the magnitude of the ischemic area. This knowledge may contribute to our mechanistic understanding of the involvement of the innate immune system during STEMI and may yield diagnostic and prognostic value for patients with myocardial infarction.