Natural Killer Cells and CD4 + T-Cells Modulate Collateral Artery Development

【摘要】  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 281-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.

The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis). Here, we show that both natural killer cells and CD4 + T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

【关键词】  peripheral vascular disease angiogenesis animal models of human disease

Introduction

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Collateral artery development (arteriogenesis) is crucial for prevention and recovery of tissue ischemia caused by arterial occlusive disease. 1 Most patients with arterial obstructions in lower extremities have well developed collateral arteries resulting in mild or no ischemic symptoms at all. Furthermore, it is a well known clinical observation that exercise improves walking distance, possibly by stimulating collateral blood flow. 2 Some patients, however, show impaired collateral formation, resulting in disabling claudication or even tissue loss ultimately requiring limb amputation. To date, it is unknown why individual differences in arteriogenesis occur. Because there is an extensive variability of the immune system within human populations, 3,4 and evidence is accumulating that immune responses play a crucial role in arteriogenesis, we here focus on cellular components of the immune system and their effects on arteriogenesis.

Several studies have implicated a role of the innate immune system by showing a function of monocytes in collateral formation. 5 In addition, it was shown that arteriogenesis is hampered in T-lymphocyte–deficient nude mice 6 and CD4 –/– mice, 7 suggesting a role of the adaptive immune system in arteriogenesis, possibly involving CD4 + T-helper cells. CD4 + T-helper cells are active in secreting cytokines and in modulating trafficking of other inflammatory cells, eg, monocytes/macrophages. 8 Several cytokines that are produced by lymphocytes, such as interleukin (IL) 10, 12, and 18, play a role in arteriogenesis. 9–11 Furthermore, expression of multiple inflammatory genes, including lymphocyte-related markers and cytokines, is elevated in a murine ischemic hindlimb model. 12 Together, these findings imply that lymphocytes contribute to arteriogenesis. However, which specific subsets of lymphocytes play a role is unknown.

Subsets of lymphocytes, such as T-cells, but also Natural Killer (NK) cells and Natural Killer T (NKT) cells, have been suggested to play a role in vascular remodeling, for example in remodeling of fetal blood supply during pregnancy, 13 or in arteriosclerosis. 14–16 Recently, it was proposed that inflammatory responses involved in plaque progression also contribute to arteriogenesis. 17 However, to our knowledge, a role of either NK-cells or NKT-cells in arteriogenesis has not been previously reported.

NK-cells, a component of the innate immune system, mediate cellular cytotoxicity among other activities and produce chemokines and inflammatory cytokines such as interferon (INF)- and tumor necrosis factor (TNF). 18 They are important in attacking pathogen-infected cells, especially during early phases of an infection. 18,19 Furthermore, NK-cells exert an immunoregulatory effect, for example via crosstalk with dendritic cells. 20

NKT-cells are a population of NK1.1 + T-cells that share some characteristics with NK-cells. Key features of NKT-cells include heavily biased T-cell receptor gene usage, CD1d restriction, and high levels of cytokine production, particularly IL-4 and INF-. 21 NKT-cells have been suggested to act as a bridge between innate and adaptive immunity. 22

Here, the roles of CD4 + T-helper cells, NK-cells, and type I NKT-cells in arteriogenesis were compared. We show for the first time that NK-cells play a role in collateral formation. In addition, we show that CD4 + T-helper cells are involved. There was no evidence for a role of type I NKT-cells in arteriogenesis.

Materials and Methods

Mouse Models

Experiments were approved by the local committee on animal welfare (TNO). C57BL/6 mice (TNO), BALB/c, and C57BL/6xBALB/c F1 mice (Harlan), Major Histocompatibility Complex (MHC) Class II–deficient mice (Taconic Farms), J 281 –/– mice (Chiba, Japan) and NK-cell–deficient mice were used. Depletion studies were performed in C57BL/6 mice using anti-CD4 (GK1.5) and anti-NK1.1 (PK136) antibody.

Surgical Procedure and Analysis of Arteriogenesis

Surgical induction of hindlimb ischemia, as well as analysis of collateral formation by either angiography or laser-doppler perfusion imaging, and tissue collection were performed as described. 23

Immunohistochemistry

Paraffin-embedded sections were stained using antibodies for CD3 (Serotec), CD4 (Abcam), NK1.1 (PK136), CD45 and MAC3 (BD Pharmingen), and PCNA (Calbiochem). The number of positively-stained cells per microscopic field was scored in a single-blinded fashion.

Real-Time RT-PCR

For TNF, TLR-4, MCP-1, GM-colony stimulating factor (CSF), and the housekeeping gene HPRT, primer-probe sets were designed using Primer Express software (Perkin Elmer). TaqMan gene expression assays for IL-1β, IL-4, IL-6, and IFN were purchased (Applied Biosystems).

Statistical Analysis

Statistics were performed as appropriate. A nonlinear mixed model was used to analyze perfusion data. For a detailed account of methodologies used see http://atvb.ahajournals.org.

Results

Mouse Strains With Different Bias in Immune Responsiveness Show a Different Ability to Develop Collateral Arteries

Lymphocyte-mediated immune responses differ between C57BL/6 and BALB/c mouse strain. 24–27 We hypothesized that this could be associated with a different ability to grow collateral arteries. To test this, unilateral femoral artery occlusion was performed in both C57BL/6 and BALB/c mice. Indeed, severe foot necrosis was observed after surgery in most BALB/c mice, whereas only sporadic necrosis of toes was observed in C57BL/6 mice. Correspondingly, collateral formation was severely impaired in BALB/c mice, whereas C57BL/6 mice demonstrated rapid arteriogenesis with functional collaterals already present at 3 to 7 days after surgery ( Figure 1 A). Quantification of angiographic images showed a significant decrease of angiographic Rentrop score in BALB/c versus C57BL/6 mice at 7 and 14 days after surgery (Rentrop score 1.6±0.3 versus 2.4±0.2 [ P =0.02, n=] and 2.2±0.3 versus 3.0±0 [ P =0.01, n=6], respectively; Figure 1 B). In addition, diameters of arterioles in adductor muscle were decreased in BALB/c mice ( Figure 1 C). Correspondingly, ischemic to nonischemic paw perfusion ratios were markedly decreased in BALB/c as compared with C57BL/6 mice throughout the observation period ( P <0.01 at all time points, n=5; Figure 1 D). In contrast, in C57BL/6 x BALB/c F1 mice perfusion recovery after surgery was as rapid as in the C57BL/6 parent strain, indicating a dominant effect of C57BL/6-genes on arteriogenesis (n=5; Figure 1 D). The 3 groups differed in rate of perfusion recovery according to a nonlinear mixed model (fixed effects: –1.900±0.143 [BL/6], –3.863±0.126 and –1.205±0.231 [F1], P <0.001 [BL/6 versus BALB/c], P <0.001 [F1 versus BALB/c], P =0.005 [F1 versus BL/6]; Figure 1 D).

Figure 1. A, Angiographic image of upper hindlimb at 7 days in C57BL/6 and BALB/c mouse. Arteriogenesis was severely hampered in BALB/c. B, Quantification of angiographic collaterals (Rentrop score; n=4 to 9). * P <0.05. C, Quantification of arteriole diameter in adductor muscle stained for -smooth muscle cells (* P <0.05, ** P <0.01, n=3). D, Ischemic/nonischemic paw perfusion ratios. As compared with C57BL/6 mice, perfusion recovery was markedly decreased in BALB/c mice, however, similar in C57BL/6xBALB/c F1 hybrid mice (n=5). * P <0.05, ** P <0.01 (black=as compared with C57BL/6, gray=as compared with F1), trend lines according to nonlinear mixed model.

Local Lymphocyte Accumulation Accompanies Development of Collateral Arteries

To study whether subsets of lymphocytes accumulate differently between C57BL/6 and BALB/c strains in areas where collaterals are formed, immunohistochemistry for both CD3 and CD4 was performed in upper limbs after unilateral femoral artery occlusion (n=3). Immunohistochemistry for NK1.1 was only performed in C57BL/6 mice, because BALB/c mice do not express the NK1.1-marker. Cells positively staining for CD3, CD4, or NK1.1 were present in adventitias of collateral arteries and increased in number over time after surgery (supplemental Figure IA through IC, available online at http://atvb.ahajournals.org). In nonoperated contralateral limbs very few lymphocytes were detected. Moreover, C57BL/6 mice accumulated significantly more lymphocytes than BALB/c mice at various time points (supplemental Figure IA and IB). Gene expressions of several lymphocyte-related cytokines were significantly different between strains, as studied in adductor muscle tissues by real-time RT-PCR; expressions were increased for IL-1β, IL-4, INF, and GM-CSF, whereas decreased for IL-6 in C57BL/6 as compared with BALB/c mice (supplemental Table II).

Impaired Collateral Formation in the Absence of CD4 + T-Lymphocytes

To more specifically determine which types of lymphocytes are involved in arteriogenesis, first the role of CD4 + T-lymphocytes was assessed. Femoral artery occlusion was performed in C57BL/6 mice depleted for CD4 + cells by anti-CD4 antibodies. Percentage of CD4 + CD3 + cells in peripheral blood was markedly reduced in CD4-depleted mice in comparison with control mice, as determined by flow cytometric analysis on the day of surgery ( P <0.01, n=5; Figure 2 A). This indicated successful depletion of CD4 + cells. Collateral formation was impaired in CD4-depleted mice as compared with control mice at 7 days (Rentrop score 2.2±0.4 versus 3.0±0.0, respectively, P =0.03; Figure 2B and 2 C). Furthermore, perfusion recovery was decreased at 7 days in anti-CD4 antibody-treated mice ( P =0.06; Figure 2 D). Rate of perfusion recovery was decreased in CD4-depleted mice according to a nonlinear mixed model (fixed effects: –1.766±0.361 and –0.998±0.273 [anti-CD4], P =0.045; Figure 2 D). These data indicate involvement of CD4 + cells in arteriogenesis.

Figure 2. A, Absence of CD4 + cells of gated CD3 + cells in peripheral blood of CD4-depleted C57BL/6 mice, but not in control mice, as indicated by flow cytometry at day of surgery. B, Angiographic image of upper hindlimb at 7 days in CD4-depleted or control mice. Collateral arteries (arrows) were less developed in CD4-depleted mice. C, Quantification of angiographic collaterals at 7 days (Rentrop score) (n=5). * P =0.03. D, Ischemic/nonischemic paw perfusion ratios. Perfusion recovery was decreased in CD4-depleted mice at 7 days (n=5). P =0.06, trend lines according to nonlinear mixed model.

Subsequently, femoral artery occlusion was performed in MHC-class-II–deficient mice, which selectively lack mature peripheral CD4 + T-helper cells. 28 Laser-doppler paw perfusion recovery was significantly decreased throughout the observation period of 4 weeks after surgery in MHC-class-II–deficient mice as compared with its C57BL/6 littermates, indicating involvement of CD4 + T-helper cells in arteriogenesis ( P <0.05 at all time points, n=10) ( Figure 3A and 3 B). Rate of perfusion recovery was decreased in MHC-class-II–deficient mice according to a nonlinear mixed model (fixed effects: –1.150±0.160 [BL/6WT] and –1.861±0.258 [MHCII –/– ], P =0.010; Figure 3 B).

Figure 3. A, Laser-doppler perfusion image of paws at 7 days after left femoral artery occlusion (arrow indicates ischemic paw) in C57BL/6 wild-type and MHC-class-II-deficient mouse. Perfusion recovery was decreased in MHC-class-II–deficient mice. B, Ischemic/nonischemic paw perfusion ratios (n=10). * P <0.05, ** P <0.01, trend lines according to nonlinear mixed model.

To exclude the possibility that the lack of lymphocytes in our model compromised the clearance of debris from ischemic tissues and fostered inflammation, thereby indirectly influencing perfusion recovery, ischemic muscle was stained for CD45, MAC3, and PCNA, as a pan-leukocyte marker, macrophage marker, and proliferation marker, respectively. Morphological signs of ischemia were similar in MHC-class-II–deficient mice (n=6) and control mice (n=4) 3 days after femoral artery occlusion. Furthermore, the number of positive cells stained for all markers was not significantly different between ischemic muscle regions of MHC-class-II–deficient mice and ischemic muscle of control mice (supplemental Figure II). These data indicate that the lack of lymphocytes does not cause by itself inflammation of ischemic tissues.

Impaired Arteriogenesis in the Absence of NK1.1-Positive Cells

Because in C57BL/6 mice collateral arteries developed much faster than the anticipated time required to generate a CD4 + T-cell response, the role of NK-cells in arteriogenesis was assessed, as NK-cells are known as rapidly reacting lymphocytes. 18,19 For this, femoral artery occlusion was performed in C57BL/6 mice treated with the NK-cell–depleting antibody anti-NK1.1, or control antibody. Percentage of NK1.1 + cells in peripheral blood was markedly reduced in NK1.1-depleted mice, as determined by flow cytometric analysis on the day of surgery ( P <0.01, n=6; Figure 4 A). A sustained reduction in NK1.1 + cells was revealed by flow cytometric analysis 14 days after surgery (data not shown). Rate of perfusion recovery was decreased in NK1.1-depleted mice as compared with control according to a nonlinear mixed model (fixed effects: –1.438±0.184 and –2.125±0.178 [anti-NK1.1], P =0.004; Figure 4 C). Moreover, comparisons at each time point showed that perfusion recovery was significantly decreased in NK1.1-depleted mice at 3 and 7 days after surgery, but was comparable to controls at 14 days and thereafter ( P =0.005, 0.009, and 0.44, respectively, n=6; Figure 4B and 4 C). Therefore, to visualize collateral formation, angiography was performed already 3 days after surgery. At this time point, collateral growth was indeed significantly impaired in NK1.1-depleted mice as compared with control (Angiographic score 0.7±0.3 versus 1.8±0.5, respectively, P =0.04, n=6; Figure 4 D). Together, these data reveal a role for NK1.1 + cells in arteriogenesis, which seemed to be most dominant in early phases of collateral growth.

Figure 4. A, Absence of NK1.1 + cells in peripheral blood of NK1.1-depleted mice, but not in control mice, as indicated by flow cytometry for both NK1.1 ( y axis) and NKG2A/C/E ( x axis) at day of surgery. B, Laser-doppler perfusion images of paws 3 days after left femoral artery occlusion (arrow indicates ischemic paw). C, Ischemic/nonischemic paw perfusion ratios (n=6). ** P <0.01. In NK1.1-depleted mice, perfusion recovery was significantly decreased in the first 7 days after femoral artery occlusion. Trend lines according to nonlinear mixed model. D, Quantification of angiographic collaterals at 3 days (Rentrop score; n=6). * P =0.04. Arteriogenesis was markedly impaired in NK1.1-depleted mice. E, Perfusion recovery was significantly decreased in NK1.1 antibody-treated MHC-class-II–deficient mice as compared with untreated MHC-class-II–deficient mice (n=4). * P <0.05, ** P <0.01 (black=as compared with MHC-class-II–deficient mice, gray=as compared with C57BL/6WT mice), trend lines according to nonlinear mixed model.

Additional Impairment of Arteriogenesis in CD4 + T-Lymphocyte-Deficient Mice After NK1.1 Depletion

To study whether NK1.1 + cells and MHC-class-II–restricted CD4 + T-cells exert their effects independently, we wished to study the effect of the absence of NK1.1 + cells on arteriogenesis in mice lacking peripheral CD4 + T-helper cells. Therefore, femoral artery occlusion was performed in MHC-class-II–deficient mice treated with NK1.1 antibodies. Perfusion recovery was markedly decreased in NK1.1 antibody-treated MHC-class-II–deficient mice as compared with nontreated MHC-class-II–deficient mice at 3 and 7 days ( P =0.07 and 0.01, respectively, n=4; Figure 4 E). Moreover, at 7 days, perfusion recovery was decreased by 55% in NK1.1 antibody-treated MHC-class-II–deficient mice as compared with its control C57BL/6 littermates, whereas MHC-class-II–deficient or NK1.1-depleted mice showed a decrease of 22% or 20% as compared with their respective controls. Overall, the 3 groups differed in rate of perfusion recovery according to a nonlinear mixed model (fixed effects: –1.246± 0.125 [BL/6WT], –1.952±0.222 [MHC II –/– ], and –2.354± 0.161 [MHCII –/–+ anti-NK1.1]; P =0.003 [BL/6WT versus MHCII –/– ], P <0.001 [BL/6WT versus MHCII –/–+ Anti-NK1.1], P =0.071 [MHCII –/– versus MHCII –/–+ Anti-NK1.1]; Figure 4 E). These findings indicate independent roles for NK1.1 + cells and MHC-class-II–restricted CD4 + T-cells in arteriogenesis.

Impaired Arteriogenesis in NK-Cell–Deficient Mice, But Not in Mice That Lack NKT-Cells

The studies described above clearly point to an independent role for CD4 + T-helper cells and NK1.1 + cells in arteriogenesis, but do not address the question whether NK1.1-positive NK-cells or NKT-cells are involved in collateral growth. The latter cells also express NK1.1 21 and are present in mice deficient for MHC class II as they are restricted by CD1d-molecules. To further define the cell type involved in promoting collateral formation, we therefore wished to study arteriogenesis in mice that selectively lack either NKT-cells or NK-cells. For this, first, femoral artery occlusion was performed in J 281 –/– mice, which have a selective loss of V 14 NKT-cells, leaving other lymphocytes, including NK-cells, intact. 29 Collateral arteries were well developed in J 281 –/– mice at 7 days ( Figure 5 A). Angiographic score was not significantly different as compared with control C57BL/6 wild-type mice (n=5; Figure 5 B). There was no significant difference in paw perfusion recovery in time after surgery between J 281 –/– and control mice (n=7, fixed effects according to nonlinear mixed model: –1.520±0.158 [BL/6WT], –1.540±0.214 [J 281 –/– ], P =0.470), indicating that type I NKT-cells are not crucial for arteriogenesis ( Figure 5 C). Second, femoral artery occlusion was performed in transgenic mice with a profound and selective deficiency in NK-cells and defective natural killing. 30 Perfusion recovery was impaired in NK-cell–deficient mice at most time points (n=8, fixed effects according to nonlinear mixed model: –0.992±0.222 [BL/6WT], –1.535±0.178 , P =0.028; Figure 5 D). These findings identify involvement of NK-cells, but not type I NKT-cells in arteriogenesis. Together, these data indicate that the above-described effects of either CD4 or NK1.1 depletion on arteriogenesis were the results of depletion of T-cells or NK-cells, respectively, but not type I NKT-cells.

Figure 5. A, Angiographic image of upper hindlimb at 7 days in C57BL/6 wild-type or J 281 –/– mouse, selectively lacking type I NKT-cells. Arteriogenesis was unaffected in J 281 –/– mice. B, Quantification of angiographic collaterals at 7 days (Rentrop score; n=5). C, Ischemic/nonischemic paw perfusion ratios (n=7). Perfusion recovery was unaffected in J 281 –/– mice as compared with C57BL/6 wild-type mice. Trend lines according to nonlinear mixed model. D, Perfusion recovery was impaired in NK-cell–deficient mice (n=8). * P <0.05, ** P <0.01, P <0.07, trend lines according to nonlinear mixed model.

Discussion

We here provide evidence for a role of both NK-cells and MHC-class-II–restricted CD4 + T-cells in arteriogenesis in mice with acute hindlimb ischemia.

Several studies have recently indicated that monocytes/macrophages play a crucial role in arteriogenesis. 5 However, roles for other inflammatory cell types in arteriogenesis have only scarcely been reported. Recently, it was shown that collateral formation is hampered in CD4 –/– mice. 7 Although CD4 can also be expressed by monocytes, NKT-cells, or dendritic cells, these data suggest a role for CD4 + T-helper lymphocytes on arteriogenesis. The purpose of the present study was to determine in more detail the role of specific subsets of lymphocytes in arteriogenesis. For illustration of the hypothetical interaction of the lymphocyte subtypes studied with respect to the initiation of arteriogenesis see supplemental Figure III.

We first studied the role of the immune system in arteriogenesis using C57BL/6 and BALB/c strains that have different genetic backgrounds and display different bias in lymphocyte-mediated immune responses. For instance, it was shown that on infection CD4 + T-cells of C57BL/6 and BALB/c mice show distinct cytokine patterns, being a T-helper 1 or T-helper 2 response, respectively. 24 Furthermore, NK-cells 25,26 and NKT-cells 27 may also react differently in C57BL/6 as compared with BALB/c mice. Our data clearly show that arteriogenesis is markedly reduced in BALB/c as compared with C57BL/6 mice. This was paralleled by decreased accumulation of lymphocytes and changed expression profile of innate cytokines around collaterals in BALB/c mice, suggesting a role of lymphocytes in arteriogenesis. It should be noted, however, that a better developed preexisting collateral network in C57BL/6 mice as compared with BALB/c mice 31 may partly explain the rapid arteriogenesis in C57BL/6 mice. In line with this, we observed a 1.3-fold increase in arteriole diameter in adductor muscle before femoral artery occlusion and increased perfusion recovery immediately after surgery in C57BL/6 as compared with BALB/c mice. Nevertheless, we here provide evidence that other, immune-related factors play an additional role in the differences in arteriogenesis between both strains.

Subsequently, we specified which subsets of lymphocytes are involved in arteriogenesis. Our data clearly point to a role of MHC-class-II–restricted CD4 + T-cells. Although this study did not address the phenotype of this CD4 + T-cell, it is tempting to speculate that CD25 + CD4 + regulating T-cells are participating. These CD4 + CD25 + regulating T-cells recognize, in contrast to "conventional" CD4 + T-cells (ie, Th1- and Th2 cells), self-antigens and are involved in maintenance of normal tissue homeostasis after insult. Moreover, they express an activated phenotype as also exemplified by expression of the activation marker CD25, indicating that they can respond swiftly to disruption of anatomic integrity. Furthermore, although arteriogenesis was not affected in J 281 –/– mice, indicating that type I NKT-cells are not involved in this process, our data cannot exclude a role for type II NKT-cells. These T-cells are CD1d-restricted but J 281-negative and thus still present in J 281 –/– mice. More detailed analysis of this cell type could be of interest as they could be responsible, like CD4 + CD25 + regulatory T-cells, for the influx of CD4 + CD3 + cells around collaterals relatively rapid after femoral artery occlusion. Nonetheless, the observed effects on the time course of perfusion recovery in the absence of NK- or T-cells suggest that NK1.1-positive cells play a role in the initiation of arteriogenesis, whereas T-cells play a role throughout the whole process. The early action of NK-cells in arteriogenesis is in accordance with the fact that NK-cells are rapidly reacting inflammatory cells, providing the first line of defense against invading pathogens. 18,19 NK-cell activation is regulated by activating and inhibitory receptors. 32 An important activating NK-receptor, NKG2D, is expressed by all NK-cells. 33 Its ligands, eg, RAE-1 molecules in mice or MHC-Class-I-related (MIC) molecules in man, are expressed on cellular stress. 34 Furthermore, MHC-class-I antigen expression is upregulated under ischemic conditions in animal models. 35 Therefore, it is tempting to hypothesize that cellular stress, induced by ischemia or elevated shear-stress in collaterals on arterial occlusion, upregulates RAE-1/MIC or MIC-like molecules. Subsequent triggering of NKG2D may lead to NK-cell activation and thereby release of inflammatory factors that are involved in arteriogenesis. Interestingly, poor collateral formation as observed in BALB/c mice was completely abolished in C57BL/6xBALB/c F1 mice that carry alleles of both C57BL/6 and BALB/c strain, indicating a dominant genetic effect of the C57BL/6 genome. One possible explanation may be that a modulated NK-receptor-ligand interaction, as reported in BALB/c mice, 26 leads to poor arteriogenesis by inadequate NK-cell activation. Receiving NK-receptor-ligand–related genes from the C57BL/6 background may reverse this deleterious effect.

Another explanation may contribute particularly to the effect of NK-cells on arteriogenesis as well. Differences in preexisting collateral networks may be genetically determined, 31,36 which may explain why one patient forms an adequate collateral network or responds well to arteriogenic treatment and the other patient does not. Interestingly, leukocytes were recently proposed to play a role in retinal vascular remodeling or pruning during development. 37 A role for the immune system in embryonic development of a collateral network is yet to be determined, but it presents a challenging additional hypothesis for the contribution of NK-cells in arteriogenesis.

We can conclude that both NK-lymphocytes and CD4 + T-lymphocytes are involved in collateral formation in mice, because the absence of these subpopulations of lymphocytes interferes with arteriogenesis. However, it should be taken into account that the evidence obtained in our mouse models is mainly indirect evidence for the proposed therapeutic beneficial use of lymphocytes for inducing arteriogenesis. Future research will determine whether and how lymphocytes play a role in arteriogenesis in man. Stimulation of arteriogenesis by specific activation of defined lymphocyte subsets might be a promising treatment for patients with ischemic disease.

Acknowledgments

We acknowledge Dave Roelen (LUMC) for helping with fluorescence-activated cell sorter analysis, Jeroen van Bergen (LUMC) for reviewing the manuscript, and Dr Wayne Yokoyama (WUSM, St. Louis, USA) and Dr Masaru Taniguchi (Chiba University, Japan) for providing us with the transgenic mice.

Sources of funding

This study was sponsored by the TNO-LUMC-VUMC tripartite angiogenesis program, the NHS molecular cardiology program (M93.001), the EU European Vascular Genomics Network (LSHM-CT-2003-503254), and DTPE grant VGT6747.

Disclosures

None.

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作者单位：Gaubius Laboratory (V.v.W., L.S., M.M.L.D., M.R.d.V., J.S., A.S., D.E., P.H.A.Q.), TNO Quality of Life, Leiden, the Netherlands; and the Departments Surgery (V.v.W., A.S., D.E., J.H.v.B., P.H.A.Q.), Rheumatology (R.E.M.T.), and Medical Statistics (P.H.E.), Leiden University Medical Center (LUMC), Le