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.

Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis.

Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa ß3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.

The relationship between coronary collateral circulation and neutrophil/lymphocyte ratio in patients with coronary chronic total occlusion.

OBJECTIVES: To investigate the relationship between neutrophil/lymphocyte ratio (NLR) and coronary collateral circulation (CCC) in patients with coronary chronic total occlusion. SUBJECTS AND METHODS: Our study population consisted of 275 consecutive patients with chronic total occlusion. One hundred and thirty-eight patients with chronic total occlusion were included in the study. They were classified into 2 groups as follows: impaired CCC (group 1: Rentrop grades 0-1) and good CCC (group 2: Rentrop grades 2-3). The NLR was calculated from the complete blood count. RESULTS: The NLR values of the patients with impaired CCC (4.5 ± 0.7) were significantly higher than of those with good CCC (2.7 ± 0.6, p < 0.001). In the multivariate logistic regression test, NLR (OR 33.36, 95% CI 8.189-135.7, p < 0.001), high-sensitivity C-reactive protein (hs-CRP; OR 2.152, 95% CI 1.226-3.777, p = 0.008), estimated glomerular filtration rate (OR 1.167, 95% CI 1.049-1.298, p = 0.004) and systolic blood pressure (OR 1.068, 95% CI 1.009-1.1310, p = 0.025) were independent predictors of impaired CCC. The NLR value >3.55 yielded an area under the curve value of 0.957 (95% CI 0.921-0.992, p < 0.001) and demonstrated a sensitivity of 95% and a specificity of 90% for the prediction of CCC. A moderate correlation between NLR and hs-CRP was observed (r = 0.443; p < 0.001). CONCLUSION: Our findings reveal that NLR correlates with the impaired development of coronary collaterals.

[Dendritic cells and coronary collateral circulation in coronary heart disease].

OBJECTIVE: To determine the relationship between the number,phenotype and functional status of dendritic cells (DCs) and coronary collateral circulation (CCC) in coronary heart disease (CHD). METHODS: Forty patients with severe coronary stenosis were recruited and divided into a CCC formation group (Group A, n=22) and a non-CCC formation group (Group B, n=18). Density gradient centrifugation was applied to separate the mononuclear cells (MNCs) from coronary artery blood samples, and MNCs were cultured and proliferated in vitro. The morphology of DCs was observed under converted microscope. The number of harvested cells and DCs was counted by hematocytometer. Flow cytometry was applied to investigate the phenotype and the mean fluorescence intensity (MFI). Mixed lymphocyte reaction was used to test the function of DCs to stimulate the proliferation of T lymphocytes. Stimulation index (SI) was calculated and compared. RESULTS: (1) After in vitro proliferation, DCs were cultured successfully from the mononuclear cells from coronary artery blood samples and the morphology of DCs was not different in the 2 groups. (2) The number of mononuclear cells (MNC no) was (3.95+/-1.41)*10(6), in the CCC group and (2.76+/-0.92)*10(6) in the non-CCC group. The MNC number was significantly increased in the CCC group (P=0.003). (3) The number of DCs was (1.54+/-0.96)*10(6) in the CCC group, and (0.99+/-0.46)*10(6) in the non-CCC group (P=0.033). (4)There was no statistical significance in the percent of CD1a+, CD1a+CD80+, CD1a+CD83+, CD1a+CD86+ cells, and MFI in the 2 groups (P>0.05). (5) SI was 4.96+/-2.30 in the CCC group, whereas 2.66+/-1.04 in the non-CCC group. The SI in the CCC group increased significantly(P=0.0003). CONCLUSION: In CHD patients with severe coronary stenosis, patients with CCC formation have higher number of DCs and stronger potential of T lymphocyte stimulation.

Natural killer cells and CD4+ T-cells modulate collateral artery development.

OBJECTIVE: The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. METHODS AND RESULTS: Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in J alpha281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II-deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. CONCLUSIONS: These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

CD8+ T lymphocytes regulate the arteriogenic response to ischemia by infiltrating the site of collateral vessel development and recruiting CD4+ mononuclear cells through the expression of interleukin-16.

BACKGROUND: Previous studies have demonstrated that macrophages and CD4+ T lymphocytes play pivotal roles in collateral development. Indirect evidence suggests that CD8+ T cells also play a role. Thus, after acute cerebral ischemia, CD8+ T cells infiltrate the perivascular space and secrete interleukin-16 (IL-16), a potent chemoattractant for monocytes and CD4+ T cells. We tested whether CD8+ T lymphocytes contribute to collateral vessel development and whether the lack of circulating CD8+ T cells prevents IL-16 expression, impairs CD4+ mononuclear cell recruitment, and reduces collateral vessel growth after femoral artery ligation in CD8(-/-) mice. METHODS AND RESULTS: After surgical excision of the femoral artery, laser Doppler perfusion imaging demonstrated reduced blood flow recovery in CD8(-/-) mice compared with C57/BL6 mice (ischemic/nonischemic limb at day 28, 0.66+/-0.04 versus 0.87+/-0.04, respectively; P<0.01). This resulted in greater calf muscle atrophy (mean fiber area, 785+/-68 versus 1067+/-69 microm2, respectively; P<0.01) and increased fibrotic tissue content (10.8+/-1.2% versus 7+/-1%, respectively; P<0.01). Moreover, CD8(-/-) mice displayed reduced IL-16 expression and decreased CD4+ T-cell recruitment at the site of collateral vessel development. Exogenous CD8+ T cells, infused into CD8(-/-) mice immediately after femoral artery ligation, selectively homed to the ischemic hind limb and expressed IL-16. The restoration of IL-16 expression resulted in significant CD4+ mononuclear cell infiltration of the ischemic limb, faster blood flow recovery, and reduced hindlimb muscle atrophy/fibrosis. When exogenous CD8+ T cells deficient in IL-16 (IL-16(-/-)) were infused into CD8(-/-) mice immediately after femoral artery ligation, they selectively homed to the ischemic hind limb but were unable to recruit CD4+ mononuclear cells and did not improve blood flow recovery. CONCLUSIONS: These results demonstrate that CD8+ T cells importantly contribute to the early phase of collateral development. After femoral artery ligation, CD8+ T cells infiltrate the site of collateral vessel growth and recruit CD4+ mononuclear cells through the expression of IL-16. Our study provides further evidence of the significant role of the immune system in modulating collateral development in response to peripheral ischemia.

Leukocyte subpopulations and arteriogenesis: specific role of monocytes, lymphocytes and granulocytes.

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.

Impaired arteriogenic response to acute hindlimb ischemia in CD4-knockout mice.

BACKGROUND: T lymphocytes, components of the immune and inflammatory systems, are involved in such normal processes as wound healing and host defense against infection and in such pathological processes as tumor growth and atherosclerotic plaque development. Angiogenesis is a mechanism common to each. Because CD4+ T lymphocytes are active in regulating humoral and cellular responses of the immune system, we determined whether CD4+ cells contribute to collateral vessel development by using the mouse ischemic hindlimb model. METHODS AND RESULTS: One week after ischemia, CD4-/- mice showed reduced collateral flow induction, macrophage number, and vascular endothelial growth factor levels in the ischemic muscle compared with wild-type mice. There was also delayed recovery of hindlimb function and increased muscle atrophy/fibrosis. Spleen-derived purified CD4+ T cells infused into CD4-/- mice selectively localized to the ischemic limb and significantly increased collateral flow as well as macrophage number and vascular endothelial growth factor levels in the ischemic muscle. Muscle function and damage also improved. CONCLUSIONS: These results indicate an important role of CD4+ cells in collateral development, as demonstrated by a 25% decrease in blood flow recovery after femoral artery ligation. Our data also suggest that CD4+ T cells control the arteriogenic response to acute hindlimb ischemia, at least in part, by recruiting macrophages to the site of active collateral artery formation, which in turn triggers the development of collaterals through the synthesis of arteriogenic cytokines.

Demostration of collaterization of the abdominal vagus in the rat using the double flourescent dyes technique [abstract]

Since the earlier report of Mitchel (1935), the central origin of preganglionic parasympathetic fibres has been studied by various investigators using different techniques and animal species. While it is now generally accepted that the dorsal motor nucleus of the vagus nerve (DMNV) is the principal source of preganglionic parasympathetic fibres to several organs in the thorax and abdomen, there has been persistent controversy as regards topographic representation of these organs in the DMNV. In a previous study in the ferret using the Horseradish peroxidase technique, some degree of topographic representation of the subdiaphragmatic part of the gastrointestinal tract was observed. It was, however, noted that no part of this nucleus is exclusively responsible for innervation of any segment of the gut. The author went on to speculate that the pattern of representation of the gut demostrated in the study might supply more than one segment of the gut by collaterization. The present study was thus designed to test this hypothesis. A total of 16 male and female Sprague Dawley rats weight range from 350 to 500g were used for the study. Four of these rats were used as control while the remaining 12 were used as experimental rats. Eight rats were injected with 1æ1 of 5 percent Diamidino yellow (DY) by multiple penetrations into the walls of the stomach while the same quantity and percentage of Fast blue (FB) was injected in the same manner into the walls of the duodenum and upper jejunum in the eight rats. Two rats had multiple injections of 1æ1 of 5 percent DY into the walls of the stomach only and two other rats had multiple injections of 1æ1 of 5 percent FB into the walls of the duodenum and upper jejunum only. Four control rats were injected with 1æ1 of normal saline (2 in the stomach and 2 in the intestine) in the same manner in which the experimental rats were injected. Each rat was anaesthetized with pentobarbitone and then perfused transcardially 14 days after the injections. Serial sections of the medulla were cut at 20-micron thickness with the cryosat and the sections examined with a Nikon Apaphot flourescence microscope. The result of the experiment revealed that in 8 rats injected with DY and FB some cells of the DMNV were labelled with DY only, some with FB only and some were doubly labelled with FB and DY. The two rats injected with FB showed FB labelled cells only while the two injected with DY showed DY labelled cells only. (AU)