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【摘要】
Objective- We analyzed the involvement of thromboxane (TX) A 2 /prostaglandin (PG) H 2 (TP) receptor in ischemia-induced neovascularization in mice.
Methods and Results- Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB 2, the stable metabolite of TXA 2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA 2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB 2 plasma levels by 2.6-fold over that of control ( P <0.01). Ang II-induced TXB 2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls ( P <0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content ( P <0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas ( P <0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II.
Conclusion- Endogenous activation of TXA 2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA 2 signaling is involved in Ang II-induced AT1-dependent vessel growth.
Endogenous activation of TXA 2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. In contrast, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effect of Ang II and fully abrogated the Ang II-induced increase in VEGF-A protein content and the number of Mac-3-positive cells.
【关键词】 angiogenesis angiotensin II ischemia thromboxane A
Introduction
Neovascularization occurs in response to local tissue ischemia mainly through inflammation and hypoxia signaling. One mechanism by which hypoxia activates blood vessel growth is the increase in hypoxia-inducible factor (HIF-1 ) leading to the expression of key growth factors, including vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF). 1 Macrophages and T lymphocytes also promote neovascularization in ischemic areas through the release of proinflammatory cytokines, production of matrix metalloproteinases, and expression of angiogenic factors. 2-4 Neovascularization takes place in the context of cross-talk involving numerous factors. Among them, hormones such as angiotensin II (Ang II) have been shown to modulate postischemic neovascularization by activation of Ang II type I receptor (AT1) and upregulation of VEGF-A signaling. 5,6 Ang II also enhances macrophage infiltration and subsequently inflammation-dependent vessel growth. 6-8
Thromboxane A 2 (TXA 2 ) is an unstable metabolite of arachidonic acid formed through the cyclooxygenase pathway. TXA 2 is released from activated platelets, monocytes, and damaged vessel wall, and causes platelet aggregation, vasoconstriction, and hypertrophy of vascular smooth muscle. 9 Action of TXA 2 is mediated by thromboxane A 2 /PG H 2 receptor (TP receptor), which are also able to bind other endogenous ligands such as endoperoxides and isoprostanes. TP receptors are widely expressed in the vasculature and exist as 2 isoforms TP and TPß in humans, whereas only TP is present in rodents. 10 As in many vasoconstrictive substances, thromboxane A2 (TXA 2 ) has the potential to participate in the regulation of blood vessel growth, but its effect on angiogenesis remain controversial. The TXA 2 mimetic IBOP inhibits migration and proliferation of cultured endothelial cells and formation of vascular-like structure in the matrigel model. 11,12 TP receptor agonists block the proangiogenic effects of FGF-2. 13 TXA 2 also abrogates VEGF-A-induced endothelial cell differentiation and migration. 14 However, TXA 2 overproduction in tumors promotes angiogenesis and tumor development, suggesting that TXA 2 is a positive regulator of blood vessel growth. 15,16 Similarly, TP blockade using the TP antagonist SQ29548 hampers VEGF-A-induced and FGF-induced endothelial cell migration and corneal neovascularization. 17,18
TXA 2 levels and TP receptor expression and activation are increased in numerous disease states, including ischemia and inflammation. 19 In addition, key angiogenic factors such as VEGF-A have been shown to activate TXA 2 signaling. 14 Recent reports also suggest that Ang II-related actions may involved activation of TXA 2 pathways. Abnormal production of TXA 2 is linked to the pathophysiology of renal and Ang II-dependent hypertension, coronary artery spasm, and arterial thrombosis. 20-22 In addition, Ang II-dependent contraction of renal afferent arterioles are mediated by TXA 2. 23 We therefore hypothesized that TXA 2 -related pathway might be activated during ischemia and may modulate either basal postischemic neovascularization or that induced by Ang II.
To this aim, we analyzed the role of basal endogenous TXA 2 signaling in spontaneous neovascularization using the TP antagonist, S 18886, in a model of surgically induced hindlimb ischemia in mice. We also determined the putative involvement of the TXA 2 -related pathway in the angiogenic effect of Ang II in this context.
Materials and Methods
Experimental Protocol
This study was conducted in accordance with both institutional guidelines and those formulated by the European community for experimental animal use (L358-86/609EEC). Male C57BL/6J mice (10 weeks old; Iffa Credo, Lyon, France; n=7 per group) were anesthetized (inhalation of isoflurane) and unilateral hindlimb ischemia was induced by right femoral artery ligature, as previously described. 3 Mice were randomly assigned to one of the following groups: (1) control group, vehicle; (2) S18886 (TP receptor antagonist, 5 mg/kg per day, (3-((6R)-6{[(4-Chlorophenyl)sulfonyl]amino}-2-methyl-5,6,7,8tetrahydro-1-naphthalenyl propanoic acid, sodium salt, drinking water; Servier), (3) S18886, (10 mg/kg per day, drinking water); (4) ramatroban (TP receptor antagonist, 10 mg/kg per day, drinking water; Bayer); (5) aspirin, (30 mg/kg per day, drinking water; Sigma); (6) Ang II (0.3 mg/kg per day, subhypertensive dose delivered by osmotic minipump); (7) Ang II (0.3 mg/kg per day) plus S18886 (10 mg/kg per day); (8) Ang II (0.3 mg/kg per day) plus ramatroban (10 mg/kg per day); (9) Ang II (0.3 mg/kg per day) plus aspirin (30 mg/kg per day); and (10) Ang II (0.3 mg/kg per day) plus candesartan (AT1 receptor blocker, 20 mg/kg per day, drinking water; Astra). Doses were chosen on the basis of previous experimental protocols in mice. 24,25 Sham-operated animals served as a control group without hindlimb ischemia.
Quantification of Neovascularization
Microangiography
After 21 days of treatment, vessel density was evaluated by high-definition microangiography using Barium sulfate (1 g/mL) injected in the abdominal aorta, followed by image acquisition with a digital X-ray transducer and computerized quantification of vessel density expressed as a percentage of pixels per image occupied by vessels in the quantification area. 3
Capillary Density
Microangiographic analysis was completed by assessment of capillary density, as previously described. 3 Ischemic and nonischemic gastrocnemius were dissected and progressively frozen in isopentane solution cooled in liquid nitrogen (LN 2 ). Sections (7 µm) were incubated with rabbit polyclonal antibody directed against total fibronectin (dilution, 1:50) to identify capillaries. Capillary density was then calculated in randomly chosen fields of a definite area using Optilab/Pro software.
Laser Doppler Perfusion Imaging
We gathered functional evidence for ischemia-induced change in mouse hindlimb vascularization. Blood perfusion in the paw was assessed through laser Doppler imaging, as previously described. 3 For each animal, perfusion was measured in both the ischemic and nonischemic paw and the ratio was calculated. Results were expressed as the mean ratio of ischemic to nonischemic values for each experimental group.
Determination of VEGF-A Expression
VEGF-A protein expression was determined by Western blot in ischemic and nonischemic legs, as previously described. 3 Gastrocnemius samples were homogenized in a lysis buffer supplemented with protease inhibitor. Samples were loaded on an SDS-PAGE gel (10%) and protein transferred to a nitrocellulose sheet (Hybond enhanced chemiluminescence ; Amersham). Blots were incubated with antibodies against VEGF-A (Santa Cruz Biotechnology; dilution of 1:2,000). As a protein loading control, membranes were stripped, incubated with a goat polyclonal antibody directed against total actin (Santa Cruz Biotechnology; dilution of 1:5000). Specific protein was detected by chemiluminescence reaction (ECL + kit; Amersham).
Determination of Plasma TXB 2 Concentration and Tissue Ang II Levels
At time of euthanization, blood was collected and plasma samples were recovered by centrifugation without clot and stored at -20°C until analysis. Plasma TXB 2 concentration was measured using a commercially available enzyme immunoassay kit (Cayman Chemical). Ang II tissue content was measured in ischemic and non ischemic gastrocnemius using Ang II enzyme immunoassay kit, according to manufacturer instructions (Spi Bio).
Evaluation of Macrophage Number (Mac-3-Positive Macrophages)
Frozen tissue sections (7 µm) were incubated with rat polyclonal antibody directed against Mac-3 (1:50; BD Pharmingen). After incubation with a biotinylated anti-rat IgG, immunostains were visualized by using avidin-biotin horseradish peroxidase visualization systems (Vectastain ABC kit elite; Vector Laboratories).
Statistical Analysis
Results are expressed as mean±SEM. One-way analysis of variance ANOVA was used to compare each parameter. Post hoc Bonferroni t test comparisons were then performed to identify which group differences account for the significant overall ANOVA. A value of P <0.05 was considered significant.
Results
Ischemia Increases Ang II and TXA 2 Plasma Levels
Plasma levels of TXB 2, the stable metabolite of TXA 2 were assayed by enzyme-linked immunosorbent assay (ELISA). Hindlimb ischemia raised TXB 2 plasma levels by 4.7-fold compared with sham nonischemic animals ( P <0.01; Figure 1 A). As expected, treatment of ischemic mice with S 18886 at 5 or 10 mg/kg per day did not significantly affect TXB 2 plasma levels. 24,25 In contrast, aspirin administration abrogated ischemia-induced upregulation of TXB 2 plasma levels ( P <0.01 versus untreated mice with hindlimb ischemia). Ischemia increases TXB 2 plasma levels, suggesting that the TXA 2 -related pathway is activated and may affect neovascularization in this setting.
Figure 1. A, Quantitative evaluation of TXB 2 content in plasma. Quantitative evaluation of microangiography (B) and foot perfusion (C) 21 days after femoral artery occlusion. Values are mean±SEM (n=7). ** P <0.01 vs sham-operated nonischemic mice. P <0.01 vs control ischemic mice. Sham indicates untreated sham-operated mice without hindlimb ischemia; Isch+ no treatment, untreated mice with hindlimb ischemia; Isch + S18886 (5), mice with hindlimb ischemia treated with S18886 at 5 mg/kg per day; Isch + S18886 (10), mice with hindlimb ischemia treated with S18886 at 10 mg/kg per day; Isch + Ram(10), mice with hindlimb ischemia treated with ramatroban at 10 mg/kg per day; and Isch + Asp, mice with hindlimb ischemia treated with aspirin at 30 mg/kg per day.
Basal TXA 2 Signaling Does Not Modulate Postischemic Neovascularization
To analyze the role of activated TXA 2 pathway in postischemic neovascularization, mice with hindlimb ischemia were treated with or without TP receptor blockers. TXA 2 pathway inhibition with aspirin (30 mg/kg per day) was the negative control. However, S 18886 or ramatroban administration did not affect the ischemic to nonischemic angiographic score ( Figure 1 B) and foot perfusion ratio ( Figure 1 C). Therefore, TXA 2 signaling is not involved in the spontaneous neovascularization in the setting of ischemia. Aspirin was also devoid of any effect on these parameters.
Involvement of TXA 2 in Ang II Proangiogenic Effect
Alternatively, TXA 2 signaling may modulate the effect of exogenously added proangiogenic factor. TXA 2 has been shown to participate in Ang II-related effects on cardiovascular homeostasis. In addition, we showed that ischemia raised Ang II tissue levels by 1.4-fold 7 days after the onset of ischemia (18.7±2.5 pg/mg proteins versus 13.4±2.1 pg/mg proteins in ischemic gastrocnemius versus non ischemic gastrocnemius; P <0.05, n=6). Exogenous administration of Ang II, by osmotic minipump, enhances Ang II tissue contents by 2.9-fold (54.2±14.2 pg/mg proteins; P <0.05 versus untreated ischemic gastrocnemius). We therefore analyzed the putative involvement of TXA 2 in Ang II proangiogenic effect in the setting of ischemia.
TXA 2 Synthesis and Ang II
In the absence of ischemia, Ang II alone increases TXB 2 plasma levels by 1.5-fold compared with untreated mice (1.9±0.3 ng/mL versus 1.3±0.2 ng/mL in Ang II-treated mice versus untreated animals, respectively; P <0.05, n=5 per group). In presence of ischemia, Ang II further enhanced TXB 2 plasma levels by 2.6-fold compared with untreated ischemic mice (12.8±2.2 ng/mL versus 4.9±0.6 ng/mL in Ang II-treated mice compared with untreated animals, respectively; P <0.01). Administration of AT1 receptor blocker reduced TXB 2 to untreated levels (5.4±1.1 ng/mL versus 12.8±2.2 ng/mL in Ang II and candesartan-treated mice compared with Ang II-treated animals; P <0.05). Treatment with S 18886 did not affect the Ang II-induced increase in TXB 2 plasma levels (12.2±1.7 ng/mL). Aspirin administration hampered the increase in TXB 2 plasma levels observed after Ang II treatment (6.8±1.0 ng/mL versus 12.8±2.2 ng/mL in Ang II-treated and aspirin-treated mice compared with Ang II-treated animals; P <0.05). Taken together, these results suggest that Ang II upregulated TXA 2 levels through AT1-dependent mechanism.
TXA 2 and Ang II Proangiogenic Effect
The angiographic score, capillary number, and paw perfusion was improved by 1.7-, 1.7-, and 1.4-fold, respectively in Ang II-treated mice when compared with controls ( P <0.05; Figure 2 ). S 18886, ramatroban, and aspirin treatments hampered the Ang II-induced increase in the neovascularization process ( P <0.05 versus Ang II-treated mice). AT1 receptor blockade also reduced Ang II-induced activation of postischemic neovascularization ( Figure 2 ).
Figure 2. Quantitative evaluation of microangiography (A), capillary density (B, capillary appears in white, arrows indicating representative examples of fibronectin-positive capillaries), and paw perfusion (C) 21 days after femoral artery occlusion. Values are mean±SEM. n=7 per group. * P <0.05 vs control ischemic mice. P <0.05 vs Ang II-treated (0.3 mg/kg per day) ischemic mice. Cont indicates untreated mice with hindlimb ischemia; Ang II, mice with hindlimb ischemia treated with Ang II; Ang II + S 18886, mice with hindlimb ischemia treated with Ang II and S18886 at 10 mg/kg per day; Ang II + Ram, mice with hindlimb ischemia treated with Ang II and ramatroban at 10 mg/kg per day; Ang II + Asp, mice with hindlimb ischemia treated with Ang II and aspirin (30 mg/kg per day); Ang II + Cand, mice with hindlimb ischemia treated with Ang II and the AT1 receptor blocker candesartan (20 mg/kg per day); Isch, ischemic leg; N.Isch, nonischemic legs.
TXA 2 and Ang II Signaling
Inflammation-Related Pathway
Treatment with Ang II enhanced the number of Mac-3-positive cells (ie, macrophages) in the ischemic hindlimbs ( P <0.05 versus control mice, n=5). Mac-3-positive cells were counted in the adventitia and perivascular space of arteries ( Figure 3 A). S 18886, ramatroban, and aspirin prevented Ang II-induced increase in macrophage infiltration. Finally, AT1 inhibition also abrogated Ang II proinflammatory-related effects. VEGF-A
Figure 3. A, Representative photomicrograph and quantitative evaluation of Mac-3-positive cells in ischemic tissue. B, Representative Western blot and quantitative evaluation of VEGF-A protein content. As a protein loading control, membranes were stripped, incubated with a goat polyclonal antibody directed against total actin. Values are mean±SEM, n=5 per group. * P <0.05 vs control ischemic mice. P <0.05 vs Ang II-treated ischemic mice. Cont indicates untreated mice with hindlimb ischemia; Ang II, mice with hindlimb ischemia treated with Ang II; Ang II + S 18886, mice with hindlimb ischemia treated with Ang II and S18886 at 10 mg/kg per day; Ang II + Ram, mice with hindlimb ischemia treated with Ang II and ramatroban at 10 mg/kg per day; Ang II + Asp, mice with hindlimb ischemia treated with Ang II and aspirin (30 mg/kg per day); Ang II + Cand, mice with hindlimb ischemia treated with Ang II and the AT1 receptor blocker candesartan (20 mg/kg per day); Isch, ischemic leg.
Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content when compared with untreated animals ( P <0.05, n=5) ( Figure 3 B). This upregulation was prevented by administration of S 18886 (10 mg/kg per day), ramatroban (10 mg/kg per day), aspirin, or AsT1 receptor blocker ( P <0.05 versus Ang II-treated mice). As a protein loading control, membranes were stripped and incubated with a goat polyclonal antibody directed against total actin ( Figure 3 B).
Discussion
The main results of this study are that TXA 2 does not modulate spontaneous neovascularization reaction after hindlimb ischemia. Conversely, TXA 2 signaling is involved in Ang II-induced AT1-dependent vessel growth in ischemic areas.
The role of TXA 2 signaling in angiogenesis remains controversial. TP receptor activation has been shown to either activate or inhibit endothelial cell proliferation and angiogenesis in different experimental models. 17,18 This apparent opposite action is likely related to the existence of 2 TP isoforms, and ß, with opposite effects. TPß expression and subsequent signaling result in inhibition of angiogenesis, whereas TP activates the angiogenic phenotype. 13,14 Animal models lack TPß, whereas cultured endothelial human cells express both isoforms and, in these cells, TPß-related effects overcome that of TP, resulting in inhibition of the angiogenic reaction. 10
In this study, we hypothesized that the TXA 2 -related pathway may affect the proangiogenic effect of exogenously added angiogenic factors, such as Ang II. In absence of ischemia, Ang II improves TXB 2 plasma levels but is unable to modulate capillary number. Similarly, normal arteries are known to be totally immune against exogenous growth factors likely because growth factor receptors are rapidly downregulated. 26,27 In contrast, in the setting of ischemia, we showed that Ang II further increases TXB 2 plasma level through AT1-dependent mechanism and that TP blockers administration hamper Ang II-induced vessel growth. In addition, TP receptor antagonists totally abrogate the Ang II-induced VEGF-A upregulation suggesting that TXA 2 is an upstream regulator of VEGF-A protein levels. S 18886 and ramatroban also reduce the number of Mac-3-positive cells in ischemic areas, demonstrating that TP receptor activation is involved in Ang II proinflammatory effect. Similarly, a specific involvement of prostanoids has been demonstrated in IL-1ß-induced angiogenesis. 24 These results are also in line with previous studies showing that TXA 2 mediates Ang II-related effects. TXA 2 triggers Ang II-dependent vasoconstriction in the kidney vasculature 23 and in rat hindlimb. 28 TXA 2 is also involved in Ang II-induced vascular smooth muscle cell (VSMC) proliferation. 29,30 Aspirin treatment abrogates Ang II-induced neovascularization, it is therefore likely that other TP receptor ligands, isoprostanes or HETE, do not interfere in this process. Taken together, our results underline that TXA 2 /TP signaling mediates Ang II proangiogenic effect in ischemic tissue.
However, TP receptor inhibition, in absence of exogenous addition of Ang II, does not modulate spontaneous neovascularization. Similarly, aspirin administration does not affect vessel growth, as previously described. 31 The reason for this discrepancy is unclear. One can first hypothesize that ischemia-induced Ang II upregulation may increase TXA 2 tissue contents to levels that are insufficient to modulate new vessel growth in ischemic areas. In support of this view, exogenous administration of Ang II markedly enhances TXB 2 plasma levels compared with untreated ischemic mice. In this context, Ang II-induced TXA 2 upregulation participates to postischemic neovascularization. Second, numerous pathways and cell types are involved in spontaneous vessel growth in vivo. Exogenous FGF enhances neovascularization in animal models of peripheral arterial occlusion, yet, the angiogenic and arteriogenic processes are unaffected in mice lacking endogenous FGF-2. 32,33 Hence, S18886 treatment alone may underestimate the importance of endogenous TXA 2 because of some sort of compensatory response by other proangiogenic pathways. Finally, TXA 2 may play a permissive role of in the proangiogenic effect of Ang II and subsequently may modulate vessel growth in pathological situations associated with Ang II level upregulation.
In conclusion, endogenous activation of TXA 2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. In contrast, TP receptor activation is involved in Ang II proangiogenic effect. This study also highlights the concept that TP receptor inhibition might be of interest in the treatment of diseases associated with Ang II overproduction, such as diabetic retinopathy.
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作者单位：Cardiovascular Research Center INSERM U689 Lariboisière (F.M., J.-S.S., L.W., M.C., M.D., B.I.L.), Université Paris, Paris, France; and the Institut de Recherches Servier (S.C., T.V., J.P.V.), Suresnes, France.Correspondance to Bernard Levy or Jean-Sebastien Silvestre, U689-INSERM, H&o