A.J.N.M. Bastiaansen

Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 Increases Neovascularization and Blood Flow Recovery After Ischemia

Rationale: Effective neovascularization is crucial for recovery after cardiovascular events. Objective: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. Methods and Results: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. Conclusions: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.

Variations in Surgical Procedures for Hind Limb Ischaemia Mouse Models Result in differences in Collateral Formation

OBJECTIVE To identify the optimal mouse model for hind limb ischaemia, which offers a therapeutic window that is large enough to detect improvements of blood flow recovery, for example, using cell therapies. MATERIALS AND METHODS Different surgical approaches were performed: single coagulation of femoral and iliac artery, total excision of femoral artery and double coagulation of femoral and iliac artery. Blood flow restoration was analysed with laser Doppler perfusion imaging (LDPI). Immuno-histochemical stainings, angiography and micro-computed tomography (CT) scans were performed for visualisation of collaterals in the mouse. RESULTS Significant differences in flow restoration were observed depending on the surgical procedure. After single coagulation, blood flow already restored 100% in 7 days, in contrast to a significant delayed flow restoration after double coagulation (54% after 28 days, P<0.001). After total excision, blood flow was 100% recovered within 28 days. Compared with total excision, double coagulation displayed more pronounced corkscrew phenotype of the vessels typical for collateral arteries on angiographs. CONCLUSION The extent of the arterial injury is associated with different patterns of perfusion restoration. The double coagulation mouse model is, in our hands, the best model for studying new therapeutic approaches as it offers a therapeutic window in which improvements can be monitored efficiently.

Lysine Acetyltransferase PCAF Is a Key Regulator of Arteriogenesis

Objective—Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP–associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. Approach and Results—After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF−/− mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6Chi monocytes, was severely impaired in PCAF−/− mice. Conclusions—These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

Galectin-2 expression is dependent on the rs7291467 polymorphism and acts as an inhibitor of arteriogenesis

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.

TLR4 Accessory Molecule RP105 (CD180) Regulates Monocyte-Driven Arteriogenesis in a Murine Hind Limb Ischemia Model

Aims We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia. Methods and Results RP105−/− and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105−/− mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105−/− mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105−/− mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105−/− mice. Conclusions RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

A limited role for regulatory T cells in post-ischemic neovascularization.

Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4+CD25+FoxP3+ T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro‐coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)‐2 with the IL‐2 monoclonal antibody JES6–1, or depleted by anti‐CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post‐ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non‐depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.

RP105 deficiency aggravates cardiac dysfunction after myocardial infarction in mice

BACKGROUND Toll-like receptor-4 (TLR4), a receptor of the innate immune system, is suggested to have detrimental effects on cardiac function after myocardial infarction (MI). RP105 (CD180) is a TLR4 homolog lacking the intracellular signaling domain that competitively inhibits TLR4-signaling. Thus, we hypothesized that RP105 deficiency, by amplifying TLR4 signaling, would lead to aggravated cardiac dysfunction after MI. METHODS AND RESULTS First, whole blood from RP105-/- and wild-type (WT) male C57Bl/6N mice was stimulated with LPS, which induced a strong inflammatory TNFα response in RP105-/- mice. Then, baseline heart function was assessed by left ventricular pressure-volume relationships which were not different between RP105-/- and WT mice. Permanent ligation of the left anterior descending coronary artery was performed to induce MI. Infarct sizes were analyzed by (immuno)histology and did not differ. Fifteen days post MI heart function was assessed and RP105-/- mice had significantly higher heart rate (+21%, P<0.01), end systolic volume index (+57%, P<0.05), end systolic pressure (+22%, P<0.05) and lower relaxation time constant tau (-12%, P<0.05), and a tendency for increased end diastolic volume index (+42%, P<0.06), compared to WT mice. In the area adjacent to the infarct zone, compared to the healthy myocardium, levels of RP105, TLR4 and the endogenous TLR4 ligand fibronectin-EDA were increased as well as the number of macrophages, however this was not different between both groups. CONCLUSION Deficiency of the endogenous TLR4 inhibitor RP105 leads to an enhanced inflammatory status and more pronounced cardiac dilatation after induction of MI, underscoring the role of the TLR4 pathway in post-infarction remodeling.

CCR7‐CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia

Background In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely “good‐responding” C57BL/6 mice and “poor‐responding” BALB/c mice. Methods and Results Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19, Ccl21a, and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7−/−/LDLR −/− mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR −/− mice. Equal numbers of α‐smooth muscle actin–positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7−/−/LDLR −/−. Fluorescence‐activated cell sorter analyses showed that numbers of CCR7+ T lymphocytes (both CD4+ and CD8+) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR −/− mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4+ T lymphocytes were decreased in the draining lymph nodes of LDLR −/− mice compared with CCR7−/−/LDLR −/− mice. Conclusions These data show that CCR7‐CCL19/CCL21 axis facilitates retention CD4+ T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis.

Toll-like receptor 4 inhibitor TAK-242 treatment does not influence perfusion recovery in tissue ischemia.

Abstract: Toll-like receptors (TLRs) are important in innate immune responses, which are crucial in collateral artery formation (arteriogenesis). TLR4−/− mice undergoing hind limb ischemia show decreased perfusion recovery accompanied by an impaired infiltration of inflammatory cells. TLR antagonists are currently developed and tested with the objective to inhibit acute exacerbation of organ damaging immune responses. However, systemic inhibition of innate immune responses may negatively influence arteriogenesis. In this study, we evaluated if TLR4 inhibition by a potent TLR4 inhibitor (TAK-242) would negatively influence perfusion recovery in a mouse model for arteriogenesis. Whole blood from human and mouse origin was stimulated with the TLR4 ligand lipopolysaccharide following TAK-242 incubation. After stimulation, cellular TLR4 activation was measured using fluorescence-activated cell sorting and tumor necrosis factor alpha release was measured using enzyme-linked immunosorbent assay. Next, the effect of TAK-242 was tested in a mouse model for arteriogenesis on perfusion recovery. TLR4 responses measured by tumor necrosis factor alpha levels were inhibited by TAK-242 in human and mouse blood after long-term stimulation. TAK-242 attenuated TLR4 responses in vivo but did not inhibit perfusion recovery in mice. In conclusion, TAK-242 does not negatively influence perfusion recovery following hind limb ischemia despite its TLR4 inhibiting properties.

The epigenetic factor PCAF regulates vascular inflammation and is essential for intimal hyperplasia development.

Objective Genetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NFκB)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia. Methods and results PCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes. To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals. Conclusions These results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.