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Background and Purpose: Oral anticoagulation prevents thromboembolism in atrial fibrillation. Factor Xa inhibitors, like edoxaban, are known to reduce inflammation and proliferation of smooth muscle cells, while vitamin K antagonism can cause vascular calcific damage. The influence of edoxaban compared to warfarin on vascular remodeling, atherosclerosis and arteriogenesis is unknown., Experimental Approach: Apolipoprotein E knockout (ApoE -/- ) mice were fed cholesterol-rich diet alone (control, co), with warfarin+vitamin K1 (warf) or with edoxaban (Edo) for 8 weeks. After 6 weeks, femoral artery ligation was performed., Key Results: There was no difference in hind-limb perfusion restoration between the three groups after 14 days (Co 0.36 ± 0.05 vs. Warf 0.39 ± 0.09 (p = .39), Co vs. Edo 0.51 ± 0.06 (p = .089), Warf vs. Edo (p = .83)) after ligation. Immuno-histologically, there was no difference in smooth muscle cell count in both hindlimbs between the three groups or in the amount of perivascular macrophages in collateral-bearing hindlimb tissue. Edoxaban showed the lowest amount of plaque tissue in the aortic sinus tissue (Co 74 ± 11% vs. Edo 62 ± 12% (p = .024), Co vs. Warf 69 ± 14% (p = .30), Edo vs. Warf (p = .14)) as well as the least amount of fibrosis (Co 3.1 ± 0.9% vs. Edo 1.7 ± 0.6% (p = .027), Co vs. Warf 4.1 ± 0.7% (p = .081), Edo vs. Warf (p < .001)). No difference in mRNA content of inflammatory cytokines in muscle tissue or spleen was detected between the three groups., Conclusion and Implications: These data suggest that treatment with edoxaban unlike warfarin prevents vascular maladaptive remodeling, which may be clinically important., Competing Interests: Declaration of Competing Interest DM has received lecture/consulting fees from Bayer and Daiichi Sankyo and research funding from Daiichi Sankyo. MB and SHS have received lecture fees and/or research funding from Boehringer Ingelheim, Bayer, Daiichi Sankyo, BMS, Pfizer, Medtronic, Abbott, Astra Zeneca, Servier, Amgen. PB and FC have no disclosures., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Coronary collateral arteries function as natural bypasses in the event of coronary obstruction. The degree of collateral network development significantly impacts the outcome of patients after an acute myocardial infarction (AMI). MicroRNAs (miRNAs, miRs) have arisen as biomarkers to identify heterogeneous patients, as well as new therapeutic targets in cardiovascular disease. We sought to identify miRNAs that are differentially expressed in chronic total occlusion (CTO) patients with well or poorly developed collateral arteries., Methods and Results: Forty-one CTO patients undergoing coronary angiography and invasive assessment of their coronary collateralization were dichotomized based on their collateral flow index (CFI). After miRNA profiling was conducted on aortic plasma, four miRNAs were selected for validation by real-time quantitative reverse transcription polymerase chain reaction in patients with low (CFI<0.39) and high (CFI>0.39) collateral artery capacity. We confirmed significantly elevated levels of miR423-5p (p<0.05), miR10b (p<0.05), miR30d (p<0.05) and miR126 (p<0.001) in patients with insufficient collateral network development. We further demonstrated that each of these miRNAs could serve as circulating biomarkers to discriminate patients with low collateral capacity (p<0.01 for each miRNA). We also determined significantly greater expression of miR30d (p<0.05) and miR126 (p<0.001) in CTO patients relative to healthy controls., Conclusion: The present study identifies differentially expressed miRNAs in patients with high versus low coronary collateral capacity. We have shown that these miRNAs can function as circulating biomarkers to discriminate between patients with insufficient or sufficient collateralization. This is the first study to identify miRNAs linked to coronary collateral vessel function in humans.

Objective: Collateral artery growth (arteriogenesis) is an important adaptive response to hampered arterial perfusion. It is unknown whether preventive physical exercise before limb ischemia can improve arteriogenesis and modulate mononuclear cell function. This study aimed at investigating the effects of endurance exercise before arterial occlusion on MNC function and collateral artery growth., Approach and Results: After 3 weeks of voluntary treadmill exercise, ligation of the right femoral artery was performed in mice. Hindlimb perfusion immediately after surgery did not differ from sedentary mice. However, previous exercise improved perfusion restoration ≤7 days after femoral artery ligation, also when exercise was stopped at ligation. This was accompanied by an accumulation of peri-collateral macrophages and increased expression of endothelial nitric oxide synthase and inducible nitric oxide synthase (iNOS) in hindlimb collateral and in MNC of blood and spleen. Systemic monocyte and macrophage depletion by liposomal clodronate but not splenectomy attenuated exercise-induced perfusion restoration, collateral artery growth, peri-collateral macrophage accumulation, and upregulation of iNOS. iNOS-deficient mice did not show exercise-induced perfusion restoration. Transplantation of bone marrow-derived MNC from iNOS-deficient mice into wild-type animals inhibited exercise-induced collateral artery growth. In contrast to sedentary controls, thrice weekly aerobic exercise training for 6 months in humans increased peripheral blood MNC iNOS expression., Conclusions: Circulating mononuclear cell-derived inducible nitric oxide is an important mediator of exercise-induced collateral artery growth., (© 2015 American Heart Association, Inc.)

Arteriogenesis as a way to restore blood flow after arterial occlusion has been under investigation for the treatment of coronary artery disease (CAD) for decades. Therapeutic approaches so far have included delivery of cytokines and growth factors as well as mechanical stimulation such as external counterpulsation. As knowledge on the mechanisms of arteriogenesis expanded, new therapeutic approaches have emerged. This review summarizes recent attempts to stimulate the growth of the coronary vasculature and discusses their potential in clinical application. This article also delivers an overview of current studies and trials on coronary arteriogenesis.

The coronary arteries have been regarded as end arteries for decades. However, there are functionally relevant anastomotic vessels, known as collateral arteries, which interconnect epicardial coronary arteries. These vessels provide an alternative source of blood supply to the myocardium in cases of occlusive coronary artery disease. The relevance of these collateral arteries is a matter of ongoing debate, but increasing evidence indicates a relevant protective role in patients with coronary artery disease. The collateral circulation can be assessed by different methods; the gold standard involves intracoronary pressure measurements. While the first clinical trials to therapeutically induce growth of collateral arteries have been unavailing, recent pilot studies using external counterpulsation or growth factors such as granulocyte colony stimulating factor (G-CSF) have shown promising results.

Objective: Lack of the adhesion molecule CD44 reduces collateral artery growth (arteriogenesis) in a murine hindlimb model. CD44 function is influenced by expression of 10 alternatively spliced exons (v1-v10), with unknown effects on arteriogenesis. As the variant exon CD44v3 binds heparan sulphate and facilitates preservation of growth factors, we hypothesized that the variably spliced exon region of CD44, especially exon CD44v3, is involved in arteriogenesis., Materials and Methods: The right femoral artery of C57BL/6J-mice was ligated and tissue was processed for histological and qPCR analysis of CD44-isoform expression. Microsphere perfusion measurements were performed in mice lacking the variably spliced exon region (CD44s knock-in mice), and in knock-in strains with specific isoform expression (CD44v3-10 and CD44v4-10), as well as in double knock-in mice, expressing CD44v3-10 and CD44s., Results: Expression of total CD44 and CD44v3 mRNA following femoral artery ligation was increased, accompanied by increased mRNA levels of the CD44-relevant splicing factors Tra2-beta1 and SRm160. CD44v3-expression was limited to the vessel wall of growing collateral arteries. Perfusion restoration was significantly reduced in mice lacking the variably spliced exon region (CD44s):20.1 ± 1.3%, compared to the background strain: 57.3 ± 2.2%. Mice expressing exon v3 (CD44v3-10) showed perfusion percentages of 25.9 ± 1.1%, compared to mice lacking this exon (CD44v4-10):19.1 ± 0.7%. Combined expression of CD44v3 and CD44s further improved perfusion restoration: 33.1 ± 2.6%., Conclusion: Total CD44 and CD44v3 mRNA are upregulated during arteriogenesis. The absence of the variably spliced exon region impairs arteriogenesis. Presence of exon v3 of CD44 results in improved arteriogenesis. Expression of CD44s and CD44v3 provides a synergistic effect on arteriogenesis. As this combined expression still resulted in hampered arteriogenesis, a specific role of exon v2 in arteriogenesis appears likely.

Aims: In patients with obstructive coronary artery disease (CAD), the growth of collateral arteries, i.e. arteriogenesis, can preserve myocardial tissue perfusion and function. Monocytes modulate this process, supplying locally the necessary growth factors and degrading enzymes. Knowledge on factors involved in human arteriogenesis is scarce. Thus, the aim of the present study is to identify targets in monocytes that are critical for arteriogenesis in patients with CAD., Methods and Results: A total of 50 patients with a chronic total coronary occlusion were dichotomized according to their collateral flow index. From each patient, RNA was isolated from unstimulated peripheral blood monocytes, monocytes stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, and from macrophages. Increased mRNA expression of galectin-2 was found in three out of four monocytic cell types of patients with a low capacity of the collateral circulation (P= 0.03 for unstimulated monocytes; P= 0.02 for LPS-stimulated monocytes; P= 0.20 for IL-4-stimulated monocytes; P= 0.02 for macrophages). Additionally, galectin-2 mRNA expression was significantly associated with the rs7291467 polymorphism in LGALS2 encoding galectin-2 in all four monocytic cell types. Patient with the rs7291467 CC genotype displayed highest galectin-2 expression, and also tended to have a lower arteriogenic response. To evaluate the effect of galectin-2 on arteriogenesis in vivo, we used a murine hindlimb model. Treatment with galectin-2 markedly impaired the perfusion restoration at Day 7., Conclusion: Collectively, these results identify galectin-2 as a novel inhibitor of arteriogenesis. Modulation of galectin-2 may constitute a new therapeutic strategy for the stimulation of arteriogenesis in patients with CAD.

Aims: Collateral arteries protect tissue from ischaemia. Heart rate correlates with vascular events in patients with arterial obstructive disease. Here, we tested the effect of heart-rate reduction (HRR) on collateral artery growth., Methods and Results: The I(f)-channel inhibitor ivabradine reduced heart rate by 11% in wild-type and 15% in apolipoprotein E (ApoE)(-/-) mice and restored endothelium-dependent relaxation in aortic rings of ApoE(-/-) mice. Microsphere perfusion and angiographies demonstrated that ivabradine did not change hindlimb perfusion in wild-type mice but improved perfusion in ApoE(-/-) mice from 40.5 ± 15.8-60.2 ± 18.5% ligated/unligated hindlimb. Heart rate reduction (13%) with metoprolol failed to improve endothelial function and perfusion. Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, and eNOS activity were increased in collateral tissue following ivabradine treatment of ApoE(-/-) mice. Co-treatment with nitric oxide-inhibitor N (G)-nitro-L-arginine methyl ester abolished the effects of ivabradine on arteriogenesis. Following ivabradine, classical inflammatory cytokine expression was lowered in ApoE(-/-) circulating mononuclear cells and in plasma, but unaltered in collateral-containing hindlimb tissue, where numbers of perivascular macrophages also remained unchanged. However, ivabradine reduced expression of anti-arteriogenic cytokines CXCL10and CXCL11 and of smooth muscle cell markers smoothelin and desmin in ApoE(-/-) hindlimb tissue. Endothelial nitric oxide synthase and inflammatory cytokine expression were unchanged in wild-type mice. Ivabradine did not affect cytokine production in HUVECs and THP1 mononuclear cells and had no effect on the membrane potential of HUVECs in patch-clamp experiments., Conclusion: Ivabradine-induced HRR stimulates adaptive collateral artery growth. Important contributing mechanisms include improved endothelial function, eNOS activity, and modulation of inflammatory cytokine gene expression.

Objectives: Our aim was to determine cytokine and oxygen gradients over the collateral circulation in humans., Background: The molecular background of the maturation of the collateral circulation in response to coronary narrowing is poorly understood in humans, partly because of difficulties in obtaining local samples from the human collateral circulation., Methods: Coronary collateral blood was sampled in 60 patients with nontotal (n = 25) or total coronary occlusions (n = 35) using a special wide-lumen catheter. Coronary collateral flow index (CFI) was assessed by intracoronary pressure measurements. Oxygenation and lactate content was measured as well as 30 cytokines potentially involved in collateral artery growth, using a custom-made multiplex assay., Results: No rise in lactate or change in pH was found in collateral blood. Oxygen gradient between coronary and collateral arterial blood correlated inversely with CFI (r = -0.61, p < 0.001). Locally increased plasma levels were found for basic fibroblast growth factor, eotaxin, macrophage migration inflammatory factor, monocyte chemoattractant protein-1, and transforming growth factor-beta, while stem cell factor and stem cell growth factor-beta were significantly decreased. The highest cytokine gradients were found in patients with the least developed collateral circulation. The majority of cytokines correlated more strongly with the pO(2) gradient across the collateral bed than with CFI., Conclusions: Intravascular ischemia during brief balloon coronary occlusion is absent in human coronary collateral arteries. The oxygen gradient found over the collateral circulation is increased in patients with a less matured collateral circulation and relates to local levels of several known pro-arteriogenic cytokines. In case of a more developed collateral circulation, relatively low levels of cytokines are present, suggesting that growth factor therapy might be beneficial at this stage.

Stimulation of collateral artery growth in patients has been hitherto unsuccessful, despite promising experimental approaches. Circulating monocytes are involved in the growth of collateral arteries, a process also referred to as arteriogenesis. Patients show a large heterogeneity in their natural arteriogenic response on arterial obstruction. We hypothesized that circulating cell transcriptomes would provide mechanistic insights and new therapeutic strategies to stimulate arteriogenesis. Collateral flow index was measured in 45 patients with single-vessel coronary artery disease, separating collateral responders (collateral flow index, >0.21) and nonresponders (collateral flow index, < or 1). Isolated monocytes were stimulated with lipopolysaccharide or taken into macrophage culture for 20 hours to mimic their phenotype during arteriogenesis. Genome-wide mRNA expression analysis revealed 244 differentially expressed genes (adjusted P, <0.05) in stimulated monocytes. Interferon (IFN)-beta and several IFN-related genes showed increased mRNA levels in 3 of 4 cellular phenotypes from nonresponders. Macrophage gene expression correlated with stimulated monocytes, whereas resting monocytes and progenitor cells did not display differential gene regulation. In vitro, IFN-beta dose-dependently inhibited smooth muscle cell proliferation. In a murine hindlimb model, perfusion measured 7 days after femoral artery ligation showed attenuated arteriogenesis in IFN-beta-treated mice compared with controls (treatment versus control: 31.5+/-1.2% versus 41.9+/-1.9% perfusion restoration, P<0.01). In conclusion, patients with differing arteriogenic response as measured with collateral flow index display differential transcriptomes of stimulated monocytes. Nonresponders show increased expression of IFN-beta and its downstream targets, and IFN-beta attenuates proliferation of smooth muscle cells in vitro and hampers arteriogenesis in mice. Inhibition of IFN-beta signaling may serve as a novel approach for the stimulation of collateral artery growth.

Objective: Circulating leukocytes play a crucial role during arteriogenesis. However, known pro-arteriogenic compounds (MCP-1, GM-CSF) acting via monocytic pathways also exert positive effects on granulocytes and lymphocytes. The role of these two cell types in arteriogenesis remains yet to be clarified, which was the aim of the current study., Methods: Ninety New Zealand White Rabbits received either phosphate buffered saline (PBS), monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) or lymphotactin (Ltn) via osmotic minipumps after unilateral femoral artery ligation. In vitro stimulation and in vivo assessment of chemoattraction confirmed cell-specific action of the compounds in rabbits. Arteriogenesis was evaluated by angiography and collateral conductance measurements using fluorescent microspheres. Quantitative immunohistology was used to quantify transmigrated leukocyte subtypes after infusion of the factors., Results: MCP-1 infusion attracts monocytes and granulocytes, whereas IL-8 attracts all three cell types albeit monocytes to a significantly lower degree than MCP-1. NAP-2 and lymphotactin selectively attract granulocytes, respectively, lymphocytes. Of the tested cytokines, only MCP-1 stimulates arteriogenesis, as assessed by collateral conductance measurements ((ml/(min 100 mmHg)): PBS, 50.70+/-5.15; MCP-1, 216.30+/-12.30; IL-8, 58.91+/-5.56; NAP-2, 66.83+/-8.72; Ltn, 52.80+/-5.37) and angiographic findings., Conclusion: This study for the first time provides evidence that not granulocytes or T-lymphocytes but monocytes are the key mediators of arteriogenesis.

Objective: Strategies to therapeutically stimulate collateral artery growth in experimental models have been studied intensively in the last decades. However, the experimental methods to detect collateral artery growth are discussed controversially and vary significantly. We compared different methods in a model of arteriogenesis in the rabbit hind limb and determined the effects on collateral flow of a known pro-arteriogenic factor, monocyte chemoattractant protein-1 (MCP-1), and a cytokine not previously evaluated for its arteriogenic efficacy, the adipocytokine leptin., Methods and Results: Forty-two New Zealand White rabbits received either MCP-1, leptin or PBS after ligation of the right femoral artery. The pro-arteriogenic effect of MCP-1 was confirmed by flow measurements during reactive hyperemia, as demonstrated by increased flow ratio (PBS 0.56+/-0.07 vs. MCP-1 0.77+/-0.06, no unit, p<0.0001), ankle-brachial index and microsphere-based conductance measurements (PBS 50.8+/-2.1 vs. MCP-1 225.8+/-8.8 ml/min/100 mm Hg, p<0.001). Biological activity of leptin on rabbit monocytes was shown by a dose dependent increase in Mac-1 expression. In-vivo administration of leptin also led to an increase in hyperemic flow and flow ratio (leptin 0.69+/-0.03, p<0.05 vs. PBS), but not to an increase in collateral conductance (leptin 54.7+/-4.1 ml/min/100 mm Hg, p=ns vs. PBS) or proliferation of vascular smooth muscle cells (Ki-67 staining: PBS 24.7+/-3.9%, leptin 22.7%+/-0.8% (p=ns), MCP-1 32.0+/-1.9% (p<0.01)). Ki-67 mRNA measured by real-time polymerase chain reaction increased (8.8+/-3.1-fold, p<0.01) during natural arteriogenesis, and was further enhanced (25.5+/-8.1-fold, p<0.005) after stimulation with MCP-1., Conclusion: MCP-1 and leptin increase collateral flow in the rabbit hind limb model. In contrast to MCP-1, leptin does not enhance direct markers of vascular proliferation such as collateral conductance under maximal vasodilation and proliferation indices. The observed increase in hyperemic collateral flow thus most probably can be attributed to the well-documented vasodilatory effects of leptin. These data stress the necessity of the use of proliferation markers and microsphere-based conductance measurements under maximal vasodilation in order to separate effects of substances on vascular proliferation from effects on vasodilation.

In the course of peripheral artery occlusive disease, blood flow to peripheral tissue progressively decreases in a substantial portion of patients, leading to insufficient oxygenation and to the occurrence of claudication or critical limb ischemia. Arteriogenesis (collateral artery growth) is a powerful natural mechanism by which large conductance vessels develop that circumvent sites of obstruction. Promising experimental data on both hypoxia-driven angiogenesis as well as monocyte-orchestrated arteriogenesis have raised high hopes for clinical application. Both endothelial growth factors to stimulate angiogenesis (i.e., capillary growth) and monocyte-attracting or -activating substances to stimulate arteriogenesis, have been proposed as potential new therapeutic agents. However, transferring the promising experimental results into clinical practice has been more cumbersome than initially anticipated. Some recent clinical studies are now focusing more specifically on the stimulation of arteriogenesis. This review will critically evaluate the results of preclinical and clinical investigations on the stimulation of vascular growth, focusing specifically on the peripheral circulation.

Peripheral arterial disease (PAD) affects a large percentage of the elderly population. Standard invasive treatment, apart from risk factor modulation, consists of bypass surgery or percutaneous transluminal angioplasty. However, symptomatic recurrence rates are high for both procedures and a substantial part of the patient population with PAD is not a candidate for invasive revascularization due to complexity of the lesion and/or co-morbidity. Therapeutic arteriogenesis has been proposed as an alternative treatment option. The present paper describes the design of the START-trial. This trial aims to determine the potential of the pro-arteriogenic substance granulocyte/macrophage colony stimulating factor (GM-CSF) to increase maximal walking distance in patients with intermittent claudication. A double-blinded, randomized, placebo-controlled study will be performed in 40 patients with peripheral obstructive arterial disease Rutherford grade I, category 2 or 3, that are candidates for bypass surgery or percutaneous transluminal angioplasty. Based on pharmacokinetic and toxicologic studies, a dose of 10 microg/kg will be used. Patients will be treated for a period of 14 days on each consecutive day, with the last injection applied on day 12. The primary endpoint will be the change in walking distance from day 0 to day 14 as assessed by an exercise treadmill test. Secondary endpoints will be the ankle-brachial index at rest and after exercise, the pain-free walking distance and cutaneous microcirculatory alterations as assessed by laser Doppler fluxmetry. Iliac flow reserve and conductance will be measured by magnetic resonance imaging.

The article offers information on research concerning the stimulation of collateral artery growth factor and its effect on patients with single-vessel coronary artery disease. It cites that patients with differing arteriogenic response as measured with collateral flow index display differential transcriptomes of stimulated monocytes. Moreover, it found that Interferon (IFN)-β inhibited smooth muscle cell proliferation in vitro and hampers arteriogenesis in mice.

The specific antagonists of tumor necrosis factor-α (TNF-α), infliximab and etanercept, are established therapeutic agents for inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although the importance of TNF-α in chronic inflammatory diseases is well established, little is known about its implications in the cardiovascular system. Because proliferation of arteriolar connections toward functional collateral arteries (arteriogenesis) is an inflammatory-like process, we tested in vivo the hypothesis that infliximab and etanercept have antiarteriogenic actions. Sixty-three New Zealand White rabbits underwent femoral artery occlusion and received infliximab, etanercept, or vehicle according to clinical dosage regimes. After 1 wk, collateral conductance, assessed with fluorescent microspheres, revealed significant inhibition of arteriogenesis (collateral conductance): 52.4 (SD 8.1), 35.2 (SD 7.7), and 33.3 (SD 10.1) ml·min-1100 mmHg-1 with PBS, infliximab, and etanercept, respectively (P < 0.001). High-resolution angiography showed no significant differences in number of collateral arteries, but immunohistochemical analysis demonstrated a decrease in mean collateral diameter, proliferation of vascular smooth muscle cells, and reduction of leukocyte accumulation around collateral arteries in treated groups. Infliximab and etanercept bound to infiltrating leukocytes, which are important mediators of arteriogenesis. Infliximab induced monocyte apoptosis, and neither substance affected monocyte expression of the adhesion molecule Mac-1. We demonstrated that TNF-α serves as a pivotal modulator of arteriogenesis, which is attenuated by treatment with TNF-α inhibitors. Reduction of collateral conductance is most likely due to inhibition of perivascular leukocyte infiltration and subsequent lower vascular smooth muscle cell proliferation. This is the first report showing a negative influence of TNF-α inhibitors on collateral artery growth. [ABSTRACT FROM AUTHOR]