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.

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.

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.

Cellular and pharmacological targets to induce coronary arteriogenesis.

The formation of collateral vessels (arteriogenesis) to sustain perfusion in ischemic tissue is native to the body and can compensate for coronary stenosis. However, arteriogenesis is a complex process and is dependent on many different factors. Although animal studies on collateral formation and stimulation show promising data, clinical trials have failed to replicate these results. Further research to the exact mechanisms is needed in order to develop a pharmalogical stimulant. This review gives an overview of recent data in the field of arteriogenesis.

Heme oxygenase-1 is required for angiogenic function of bone marrow-derived progenitor cells: role in therapeutic revascularization.

AIMS: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. RESULTS: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin(-)CD45(-)cKit(-)Sca-1(+)VEGFR-2(+)) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1(-/-) BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1(+/-) diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1(+)CXCR-4(+)) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. INNOVATION: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. CONCLUSIONS: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.

Magnetic resonance imaging-defined areas of microvascular obstruction after acute myocardial infarction represent microvascular destruction and haemorrhage.

AIMS: Lack of gadolinium-contrast wash-in on first-pass perfusion imaging, early gadolinium-enhanced imaging, or late gadolinium-enhanced (LGE) cardiovascular magnetic resonance (CMR) imaging after revascularized ST-elevation myocardial infarction (STEMI) is commonly referred to as microvascular obstruction (MVO). Additionally, T2-weighted imaging allows for the visualization of infarct-related oedema and intramyocardial haemorrhage (IMH) within the infarction. However, the exact histopathological correlate of the contrast-devoid core and its relation to IMH is unknown. METHODS AND RESULTS: In eight Yorkshire swine, the circumflex coronary artery was occluded for 75 min by a balloon catheter. After 7 days, CMR with cine imaging, T2-weighted turbospinecho, and LGE was performed. Cardiovascular magnetic resonance images were compared with histological findings after phosphotungstic acid-haematoxylin and anti-CD31/haematoxylin staining. These findings were compared with CMR findings in 27 consecutive PCI-treated STEMI patients, using the same scanning protocol. In the porcine model, the infarct core contained extensive necrosis and erythrocyte extravasation, without intact vasculature and hence, no MVO. The surrounding-gadolinium-enhanced-area contained granulation tissue, leucocyte infiltration, and necrosis with morphological intact microvessels containing microthrombi, without erythrocyte extravasation. Areas with IMH (median size 1.92 [0.36-5.25] cm(3)) and MVO (median size 2.19 [0.40-4.58] cm(3)) showed close anatomic correlation [intraclass correlation coefficient (ICC) 0.85, r = 0.85, P = 0.03]. Of the 27 STEMI patients, 15 had IMH (median size 6.60 [2.49-9.79] cm(3)) and 16 had MVO (median size 4.31 [1.05-7.57] cm(3)). Again, IMH and MVO showed close anatomic correlation (ICC 0.87, r = 0.93, P < 0.001). CONCLUSION: The contrast-devoid core of revascularized STEMI contains extensive erythrocyte extravasation with microvascular damage. Attenuating the reperfusion-induced haemorrhage may be a novel target in future adjunctive STEMI treatment.

MicroRNA-34a regulates cardiac ageing and function.

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

Microvascular proliferations in arteriovenous malformations relate to high-flow characteristics, inflammation, and previous therapeutic embolization of the lesion.

BACKGROUND: Episodes of microvascular proliferation associated with volume expansion have been observed in arteriovenous malformations (AVMs) of skin and soft tissue. OBJECTIVE: We sought to investigate the relationship between a microvascular proliferative response and flow velocity in AVMs. METHODS: Resection specimens of 80 AVMs were clinically categorized as either high- or low-flow lesions, and histopathologically screened for the presence of microvessels, inflammation, thrombosis, or a combination of these. Immunohistochemistry was performed with endoglin (CD105), von Willebrand factor, and fibrinogen antibodies. RESULTS: Clinically, 37 AVMs were classified as high-flow lesions and 43 as low-flow lesions. In 81% of high-flow lesions microvascular proliferations were seen versus in 14% of low-flow lesions (P < .005). In high-flow lesions, which were embolized before surgery (30% of all), 88% showed microvascular proliferation, 88% inflammation, and 33% thrombosis. However, similar vasoproliferative responses were also observed in nonembolized AVM (69% high-flow and 14% low-flow lesions). Endoglin was more frequently expressed in high-flow lesions. Extracellular von Willebrand factor staining was found in most lesions, irrespective of flow type or presence of microvascular proliferations. LIMITATIONS: The study was carried out at a single tertiary referral center. CONCLUSIONS: Microvascular proliferative masses in AVMs appear to be strongly associated with high-flow characteristics. This could be explained to some extent by previous therapeutic embolization and/or inflammation in the lesion. However, occurrence of similar microvascular responses in AVM that were not embolized before surgery suggests that the biomechanical effects of high flow in these lesions may also have an angiogenic effect.

The diverse identity of angiogenic monocytes.

BACKGROUND: The role of bone marrow-derived cells in stimulating angiogenesis, vascular repair or remodelling has been well established, but the nature of the circulating angiogenic cells is still controversial. DESIGN: The existing literature on different cell types that contribute to angiogenesis in multiple pathologies, most notably ischaemic and tumour angiogenesis, is reviewed, with a focus on subtypes of angiogenic mononuclear cells and their local recruitment and activation. RESULTS: A large number of different cells of myeloid origin support angiogenesis without incorporating permanently into the newly formed vessel, which distinguishes these circulating angiogenic cells (CAC) from endothelial progenitor cells (EPC). Although CAC frequently express individual endothelial markers, they all share multiple characteristics of monocytes and only express a limited set of discriminative surface markers in the circulation. When cultured ex vivo, or surrounding the angiogenic vessel in vivo, however, many of them acquire similar additional markers, making their discrimination in situ difficult. CONCLUSION: Different subsets of monocytes show angiogenic properties, but the distinct microenvironment, in vitro or in vivo, is needed for the development of their pro-angiogenic function.

The shear stress-induced transcription factor KLF2 affects dynamics and angiopoietin-2 content of Weibel-Palade bodies.

BACKGROUND: The shear-stress induced transcription factor KLF2 has been shown to induce an atheroprotective phenotype in endothelial cells (EC) that are exposed to prolonged laminar shear. In this study we characterized the effect of the shear stress-induced transcription factor KLF2 on regulation and composition of Weibel-Palade bodies (WPBs) using peripheral blood derived ECs. METHODOLOGY AND PRINCIPAL FINDINGS: Lentiviral expression of KLF2 resulted in a 4.5 fold increase in the number of WPBs per cell when compared to mock-transduced endothelial cells. Unexpectedly, the average length of WPBs was significantly reduced: in mock-transduced endothelial cells WPBs had an average length of 1.7 µm versus 1.3 µm in KLF2 expressing cells. Expression of KLF2 abolished the perinuclear clustering of WPBs observed following stimulation with cAMP-raising agonists such as epinephrine. Immunocytochemistry revealed that WPBs of KLF2 expressing ECs were positive for IL-6 and IL-8 (after their upregulation with IL-1ß) but lacked angiopoietin-2 (Ang2), a regular component of WPBs. Stimulus-induced secretion of Ang2 in KLF2 expressing ECs was greatly reduced and IL-8 secretion was significantly lower. CONCLUSIONS AND SIGNIFICANCE: These data suggest that KLF2 expression leads to a change in size and composition of the regulated secretory compartment of endothelial cells and alters its response to physiological stimuli.