Role of T-lymphocytes in angiotensin II-mediated microvascular thrombosis

Abstract

Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Since T-lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (AngII) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during AngII-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild type (WT), immunodeficient Rag-1^−/−, CD8⁺ or CD4⁺-lymphocyte-deficient, and NADPH oxidase (gp91^phox) deficient mice implanted with an AngII-loaded pump for 2 weeks. Chronic AngII infusion enhanced arteriolar thrombosis in WT mice but not in Rag-1^−/−, CD4⁺T-cell deficient, or gp91^phox−/− mice. CD8⁺ T-cell^−/−-mice exhibited partial protection. Adoptive transfer of T-cells derived from WT- or gp91^phox−/−-mice into Rag-1^−/− restored the prothrombotic phenotype induced by AngII. T-lymphocytes (CD4⁺ and, to a lesser extent, CD8⁺) play a major role in mediating the accelerated microvascular thrombosis associated with AngII-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other T-lymphocytes, also appear critical for the AngII-enhanced, T-cell dependent thrombosis response.

Introduction

The risk factors for cardiovascular disease (CVD), including hypertension, have been shown to produce structural and functional alterations in large and microscopic blood vessels that ultimately lead to end-organ damage^1–3. While a variety of mechanisms have been proposed to explain the vascular dysfunction induced by CVD risk factors, recent attention has focused on the pro-oxidative, pro-inflammatory, and pro-thrombogenic phenotype that is assumed by the vasculature in the presence of one or more risk factors. Characteristic features of the altered vascular phenotype induced by CVD risk factors include an accumulation of leukocytes and platelets on the vessel wall (with subsequent transendothelial migration of leukocytes), increased production of reactive oxygen species (ROS) by vascular endothelium and circulating blood cells, impaired vasomotor and endothelial barrier functions, and enhanced thrombus formation¹. Since the aforementioned responses are also elicited in different acute and chronic inflammatory diseases, it has been suggested that CVD risk factors exert their deleterious effects on the vasculature through activation of the innate and/or adaptive immune systems, which have also linked to oxidative stress and hypercoagulation/thrombosis^{1, 4}.

Angiotensin II (AngII) has been implicated as a potential initiator of the inflammatory phenotype and vascular dysfunction that are associated with different CVD risk factors^1,4–6. Animal studies have revealed that acute or chronic administration of AngII induces ROS production, impairs vasomotor function^{5, 7, 8}, promotes the adhesion of leukocytes and platelets to endothelial cells lining the microvasculature⁹, and enhances thrombus formation^{10, 11}. The oxidative stress elicited by AngII has been attributed to activation of NAD(P)H oxidase, which is expressed in a variety of cells, either circulating in blood or comprising the vessel wall^{8, 12}. A role for AngII in mediating the vasomotor dysfunction and increased incidence of thrombotic events in human hypertension is supported by clinical studies showing a reversal of these responses in patients treated with angiotensin converting enzyme inhibitors or angiotensin receptor blockers¹³. Recent animal studies have revealed that the phenotypic changes in the vasculature caused by AngII are linked to activation of the adaptive immune system^{5, 14}. The elevated blood pressure, vasomotor dysfunction, and oxidative stress elicited by AngII in wild type (WT) mice are not observed in T-lymphocyte deficient Rag-1^−/− mice, while adoptive transfer of WT T-cells into Rag-1^−/− restores the AngII phenotype. The T-cell dependent vascular alterations induced by AngII also appear to be linked to NADPH oxidase activity in T-cells inasmuch as adoptive transfer of T-cells from p47^phox (a protein subunit of NADPH oxidase) deficient mice into Rag-1^−/− only partially restores the AngII phenotype^{5, 14}.

Clinical and experimental evidence implicates AngII in the genesis of thrombosis in both large and microscopic blood vessels. Angiotensin II has been shown to activate the coagulation pathway, inhibit fibrinolysis, promote platelet aggregation, and enhance the rate of thrombus development in the microvasculature^{10, 13, 15, 16}. While different cell (platelets, endothelial cells) and receptor populations (e.g., AngII type-2 and -4 receptors) have been implicated in AngII-enhanced thrombosis¹⁰, it remains unclear whether the adaptive immune system also contributes to this response. In light of growing evidence that links adaptive immunity to hemostasis¹⁷, and the known association between immune cells and other AngII-mediated vascular responses (elevated blood pressure, impaired vasomotion)⁵, a role for T-lymphocytes in AngII-mediated thrombosis appears tenable. Hence, the overall objective of this study was to test the hypothesis that T-lymphocyte-associated NADPH oxidase contributes to the accelerated microvascular thrombosis associated with AngII-induced hypertension. Our findings implicate both activated/effector/memory CD4⁺ T-lymphocytes (CD44^highCD62L^neg) and NADPH oxidase (in cells other than T-cells) in the accelerated microvascular thrombosis associated with chronically elevated AngII levels.

Materials and methods

Animals

C57BL/6 (wild-type [WT] n = 47), CD8+^−/− (B6.129S2-Cd8a^tm1Mak/J; n = 6), CD4+^−/− (B6.129S2-Cd4^tm1Mak/J; n = 6), gp91^phox−/− (B6.129S6-Cybb^tm1Din/J; n = 15) and Rag-1^−/− (B6;129S7-Rag1^tm1Mom/J; n = 29), IFNγ^−/− (B6.129S7-Ifng^tm1Ts/J; n=5) and TNFαr^−/− (B6.129-Tnfrsf1a^tm1Mak/J; n=5) mice were obtained from Jackson Laboratories at age 6–8 weeks (Bar Harbor, ME). All animal experiments were performed according to the criteria outlined by the National Institutes of Health, and were approved by the Institutional Animal Care and Use Committee of LSU Health Sciences Center.

Angiotensin II infusion

Saline- or AngII- (1 µg/kg/min) loaded micro-osmotic pumps (Alzet, Cupertino, CA, model 1002) were implanted up to 14 days subcutaneously (intrascapular region) under isofluorane anesthesia using sterile procedures, as previously described^{9, 10}. Systolic blood pressure (SBP) values were obtained using a computerized tail cuff system (Hatteras Inst, Inc.) before and during (day 14^th) AngII infusion¹⁰.

DOCA-salt-induced hypertension

To determine whether enhanced microvascular thrombosis is also evident in a low renin model of hypertension, the deoxycorticosterone acetate (DOCA)-salt model was used in WT mice (n = 16). The mice, anesthetized with ketamine (150 mg/kg, ip) and xylazine (7.5 mg/kg, ip), underwent a uninephrectomy and subcutaneous implantation of a 50 mg (21-day-release) DOCA pellet (Innovative Research of America, Sarasota, FL)) in the mid-scapular region. After surgery, the mice received the analgesic Carprofen (5 mg/Kg, s.c.) and then placed on 1% saline/0.2% KCl drinking solution for 3 weeks. Sham controls were produced by removing a kidney, but without implantation of a DOCA pellet. The sham group was placed on regular drinking water plus 0.2% potassium chloride. SBP was monitored using the tail-cuff method.

T-cell transfer

T-lymphocytes were isolated from the spleen of WT (C57BL/6) or gp91^phox−/− donor mice using a Dynal® Mouse T Cell Negative Isolation Kit- (Invitrogen Dynal AS, Oslo, Norway) and injected (^~10⁷ cells in 0.2 ml of PBS, iv) into Rag-1^−/− recipients prior to AngII pump implantation.

Cremaster muscle procedure

On the 15^th day following AngII- or saline-loaded pump implantation, or the 21^th day following uninephrectomy (both sham & DOCA-salt), the mice were anesthetized with pentobarbital (50 mg/kg, ip). The right carotid artery was cannulated for measurement of arterial blood pressure and the right jugular vein was cannulated for drug administration. The cremaster muscle was prepared for intravital microscopic observation as previously described¹⁰, and arterioles with diameters between 30–36 µm and a wall shear rate (WSR) >500 sec⁻¹ were selected for study.

Light/dye-induced thrombosis

Thrombus formation was evaluated in cremaster arterioles as previously described¹⁰. After light/dye injury (induced by activation of intravascular FITC-dextran), the arteriole under study was continuously epi-illuminated and thrombus formation was quantified by determining: 1) the time of onset of platelet deposition/aggregation within the microvessel (onset time), and 2) the time required for complete flow cessation for ≥ 30 sec (cessation time). Epi-illumination was discontinued once blood flow ceased in the vessel under study. Typically, 1 – 3 thrombi were induced in each mouse and the results of each vessel were averaged.

Flow cytometic analysis of activated T cell phenotype in peripheral blood and spleen was performed as previously described¹⁸. For a more detailed method, see the online Data Supplement (available at http://hyper.ahajournals.org).

Experimental groups

Light/dye-induced thrombus development was evaluated in arterioles of each of the following experimental groups: 1) WT mice + saline pump (n = 7), 2) WT mice + AngII pump (n = 9), 3) WT mice + uninephrectomy (sham controls) (n = 5), 4) WT + uninephrectomy + DOCA-salt (n = 6), 5) lymphocyte deficient Rag-1^−/− mice + saline (n=7) or AngII pump (n = 6), 6) Rag-1^−/− mice + AngII pump reconstituted with T-cells from WT donors (n = 6), 7) Rag-1^−/− mice + AngII pump reconstituted with T-cells from gp91^phox−/− donors (n = 4), 8) gp91^phox−/− mice + saline (n=8) or AngII pump (n = 6), 9) CD8⁺ T-cell^−/− mice + AngII pump (n = 6), 10) CD4⁺ T-cell^−/− mice + AngII pump (n = 6), 11) IFNγ^−/− +AngII pump (n=5), and 12) TNFαr^−/− +AngII pump (n=5).

Data analysis

All data are presented as mean ± SEM. Group comparisons were made using a 1-way ANOVA followed by the Newman-Keuls posthoc test. Statistical significance was set a p<0.05.

Results

AngII enhances light/dye-induced thrombosis in cremaster muscle arterioles

Figure 1 summarizes the thrombosis responses to light/dye injury in two distinct murine models of hypertension, i.e., AngII- and DOCA-salt-induced hypertension. After 2 wks of AngII infusion, systolic blood pressure (SBP) was increased to 153.8 ± 8.6 mmHg in WT mice, compared to 107 ± 2.2 mmHg in WT mice with a saline pump. Enhanced thrombus development during chronic AngII infusion is evidenced by the reductions of the onset time (thrombus initiation) and time for flow cessation (propagation/stabilization). However, mice with DOCA-salt induced hypertension (SBP, 142.7 ± 7.3 mmHg) did not exhibit an altered thrombosis response to light/dye injury, when compared to sham (uninephrectomy alone) controls.

Figure 1.

Light/dye-induced thrombus formation in cremaster muscle arterioles of wild type (WT) mice with either (Panel A) angiotensin II (AngII)- or (Panel B) DOCA-salt-induced hypertension. Control groups for the AngII model include WT mice and WT mice implanted with a saline-loaded pump (WT-saline). The DOCA-salt model included a control group exposed to uninephrectomy alone (WT-Sham). * indicates p<0.01 vs the WT-Saline group.

Lymphocyte deficiency protects against AngII-enhanced microvascular thrombosis

Figure 2 compares the effects of chronic AngII infusion on light/dye induced thrombus development in WT and lymphocyte deficient Rag-1^−/− (SBP, 158.7 ± 8.6 mm Hg) mice. Unlike in WT mice, AngII did not accelerate the rate of thrombus formation in Rag-1^−/− mice. However, following adoptive transfer of WT T-lymphocytes for a period of 2 wks in Rag-1^−/− (SBP, 173 ± 4.8 mm Hg) mice, the prothrombotic phenotype induced by AngII infusion was fully restored, which suggests that T-cells play a major role in AngII mediated thrombosis.

Figure 2.

Role of T-lymphocytes in angiotensin II-enhanced, light/dye-induced thrombus formation. WT-Saline – wild type mice implanted with saline-loaded pumps; WT-AngII – wild type mice implanted with AngII-loaded pumps; Rag-1^−/−-saline – immunodeficient Rag-1^−/− mice implanted with saline-loaded pump; Rag-1^−/−-AngII – immunodeficient Rag-1^−/− mice implanted with AngII-loaded pumps; Rag-1^−/−-AngII←WT T-cells – Rag-1^−/− mice (with AngII pumps) reconstituted with T cells obtained from WT donor mice. * indicates p<0.01 vs WT-Saline; & indicates p<0.01 vs Rag-1^−/−-AngII.

CD4⁺-T-cells, and to a lesser extent CD8⁺-T-cells, mediate the T-lymphocyte dependent enhancement of thrombosis associated with AngII-induced hypertension

In order to determine which T-lymphocyte subpopulation accounts for the T-cell dependent thrombotic response to AngII, we compared thrombus development in mice that are genetically deficient in either CD4⁺- or CD8⁺-T-cells to their WT counterparts (Figure 3). The CD4⁺-T-cell deficient mice (SBP, 170.7 ± 4.8 mmHg) responded to chronic AngII infusion in a manner similar to that noted in Rag-1^−/− mice, i.e., the acceleration of time of onset and time to flow cessation induced by AngII was completely prevented. While CD8⁺-T-cell deficient mice (SBP, 161.4 ± 11.3 mmHg) were also protected against AngII thrombosis, this was only reflected in the restored time of onset, with no improvement in the time to flow cessation, suggesting that CD8⁺-T-cell^−/− mice assume a prothrombogenic phenotype during chronic AngII infusion.

Figure 3.

Role of CD4⁺ and CD8⁺ T-lymphocytes in angiotensin II-enhanced, light/dye-induced thrombus formation. WT-Saline – wild type mice implanted with Saline-loaded pumps; WT-AngII – WT mice implanted with AngII-loaded pumps; CD8⁺ T-cell^−/−-AngII – CD8⁺ T-cell deficient mice implanted with AngII pumps; CD4⁺ T-cell^−/−-AngII - CD4⁺ lymphocyte deficient mice implanted with AngII-loaded pumps. *indicates p<0.01 vs WT-Saline; & indicates p<0.01 vs WT-AngII.

Phenotypic analysis of T cells in peripheral blood and spleen revealed an approximately 15% reduction (42 ± 2.0% vs. 49.35 ± 0.85%) in CD4⁺ T cells within TCRab subset in spleens of AngII-treated mice, compared with controls (data not shown). A 35.5% increase (17.2 ± 0.9% vs. 12.7 ± 1.2%) in percentage of activated/effector/memory CD4⁺ lymphocytes was detected in spleen, while 68% (10.4 ± 1.6% vs 6.2 ± 0.5%) more activated/effector/memory CD4⁺ cells (CD62L^negCD44^high) were measured in blood of AngII-treated mice, compared to the WT control group (Figure S1). No significant changes were noted in the activation state of CD8⁺ T-cells in both spleen and blood between control and AngII-treated mice (Figure S2).

Intracellular production of IFN-γ, IL-4 and IL-17 cytokines by splenic CD4⁺ T cells isolated from control and AngII-treated mice were not different (Figure S3). This is consistent with the observation that AngII enhanced, light/dye-induced thrombosis did not differ between IFN-γ-deficient and WT mice (Figure S4). However, partial protection against AngII-enhanced thrombosis (as reflected by an improved time of onset of thrombosis) was detected in mice deficient in TNFα receptors (Figure S4).

NADPH oxidase, associated with cells other than T-lymphocytes, contributes to AngII-accelerated thrombus development

The role of NADPH in AngII-enhanced microvascular thrombosis was evaluated using mice deficient in the critical NADPH oxidase subunit, gp91^phox−/− (SBP, 141.4 ± 6.6 mm Hg) (Figure 4, panel A) These mutant mice exhibited complete protection against the prothrombotic effects of AngII infusion, as evidenced by the normalized values for time of onset and time to flow cessation. In order to determine whether NADPH oxidase in T-lymphocytes accounts for the protection observed in gp91^phox−/− mice, adoptive transfer experiments were performed wherein T-cells derived from either WT or gp91^phox−/− mice were administered to Rag-1^−/− mice with AngII pumps (SBP, 163.3 ± 2.4 mm Hg) (Figure 5, panel B). These experiments revealed that Rag-1^−/− mice reconstituted with T-cells derived from either WT or gp91^phox−/− mice exhibited a fully restored thrombosis response to AngII, suggesting that NADPH oxidase in T-cells is not critical for AngII-induced thrombosis.

Figure 4.

Contribution of NADPH oxidase to angiotensin II-enhanced, light/dye-induced thrombus formation. WT-saline – wild type mice implanted with saline-loaded pumps; WT-AngII – WT mice implanted with AngII-loaded pumps; gp91^phox−/−-saline- gp91^phox-deficient mice implanted with saline pump; gp91^phox−/−-AngII- gp91^phox-deficient mice implanted with AngII pump (panel A); Rag-1^−/−-AngII – Rag-1^−/− mice implanted with AngII-loaded pumps; Rag-1^−/−-AngII←WT T-cells – Rag-1^−/− mice (with AngII pumps) reconstituted with T cells obtained from WT donor mice; Rag-1^−/−-AngII←gp91^phox−/− T-cells – Rag-1^−/− mice (with AngII pumps) reconstituted with T cells obtained from gp91^phox-deficient donor mice. Panel A: * p<0.01 vs WT-Saline; & denotes p < 0.05 vs WT-AngII; && denotes p <0.01 vs WT-AngII; Panel B: **p<0.01 vs Rag-1 ^−/− -AngII, * p<0.05 vs Rag-1^−/− -AngII.

Discussion

The RAS has been implicated in different characteristic responses of the vasculature to chronic arterial hypertension, including vasomotor dysfunction, inflammation, oxidative stress, and thrombogenesis^{5, 14, 19, 20}. While there is a large and growing body of evidence that suggests a causal link between the elevated blood pressure and vasomotor dysfunction induced by AngII with inflammation and oxidative stress^{5, 7}, the contributions of the latter responses to the enhanced thrombus formation associated with AngII dependent hypertension remains unclear. In the present study, we provide evidence for the involvement of the adaptive immune system as well as NADPH oxidase-dependent oxidative stress in the accelerated thrombus development that occurs in response to chronically elevated AngII levels.

We have previously reported that chronic AngII infusion in mice results in accelerated thrombus formation in the microvasculature after light/dye injury¹⁰. The AngII-mediated thrombogenic response, which is more evident in arterioles than venules, involves the activation of several receptor populations, including angiotensin (type-2 & -4), endothelin-1 (ET-1A), and bradykinin (BK-1) receptors. The elevated blood pressure that accompanies AngII infusion was considered an unlikely cause of the accelerated thrombosis because AT1 receptor blockade (or genetic deficiency) ablates the AngII-induced hypertension without altering thrombus development. The present study provides additional evidence that hypertension per se is not a major determinant of the accelerated thrombosis observed during chronic AngII infusion. A comparison of the light/dye-induced thrombosis responses between mice with DOCA-salt hypertension (a low renin model) and AngII-induced hypertension demonstrated that the accelerated thrombosis response was unique to the AngII model, despite comparable elevations in blood pressure in the two models of hypertension. The results of a previous study²¹ in a rat model of hypertension tend to support our findings. Capers and coworkers noted that AngII infusion-induced hypertension was associated with a 10-fold increase in aortic expression of thrombin receptor mRNA, while no change in mRNA levels were detected in Dahl-salt sensitive (low renin) hypertensive rats. Furthermore, they demonstrated that treatment of the AngII infused rats with superoxide dismutase prevented the upregulation of thrombin receptor mRNA, implicating oxidative stress in this response. The latter observation is interesting in view of documented evidence that both the AngII and low renin models of hypertension are associated with oxidative stress ^{5, 14}.

Recent reports have attributed a major role to T-lymphocytes in mediating the elevated blood pressure and impaired endothelium-dependent vasodilation that are associated with chronic AngII infusion^{5, 22, 23}. These reports indicate that, unlike their wild type counterparts, immunodeficient Rag-1^−/− mice do not exhibit hypertension and vasomotor dysfunction in response to elevated AngII levels, suggesting an immunological basis for the altered vascular tone and reactivity induced by chronic AngII exposure. The results of the present study suggest that the adaptive immune system also contributes to the enhanced thrombogenesis induced by AngII, with CD4⁺ T-cells and, to a lesser extent, CD8⁺ T-cells mediating this response. Our conclusions regarding the involvement of T-lymphocytes in AngII-enhanced thrombogenesis is based on 4 lines of evidence: 1) absence of an accelerated thrombosis response to AngII in immunodeficient Rag-1^−/− mice, 2) restoration of the AngII-mediated thrombosis in Rag-1^−/− mice following adoptive transfer of T-cells derived from WT mice, 3) absence of AngII-enhanced thrombosis in mice that are genetically deficient in CD4⁺ T-cells, with a partial reduction noted in CD8⁺ T-cell deficient mice, and 4) the activation status of CD4⁺ T cells in the spleens and peripheral blood of AngII-treated mice. The larger number of activated CD4⁺ T-cells in AngII-treated mice is consistent with previous reports⁵. The accumulation of activated/effector/memory CD4⁺ T-cells in spleen is consistent with CD4⁺ T cell recruitment in peripheral organs^{5, 24–26} of AngII-treated mice, compared to the WT controls. The interesting dual contribution of CD4⁺ and CD8⁺ T lymphocytes to the initiation phase (onset) of thrombus development is consistent with cross-talk between the two T-cell populations ^27,28.

The mechanism(s) that underlie the ability of the T-cell to accelerate microvascular thrombosis in the presence of AngII remain unclear. T-cells, nevertheless, are known to directly interact and communicate with platelets^{29, 30, 31}, release cytokines that can activate coagulation cascade (e.g., induce tissue factor) and promote thrombus formation^{29, 32, 33}. Chronic AngII infusion is associated with increased plasma levels of IL-6^{34, 35} and an increased production of TNF-α by T-lymphocytes⁵. Furthermore, T-cell derived cytokines such as TNF-α and IL-1β have been shown to accelerate light/dye induced thrombus formation in arterioles^{36, 37}. Our negative findings in AngII-treated IFN-γ deficient mice suggest that this cytokine is an unlikely mediator of this T-cell dependent thrombosis response. However, we did detect partial protection against AngII-induced thrombosis in TNF-α receptor deficient mice, which implicates at least a small role for this cytokine. Interleukin-6, which has been implicated in the pathogenesis of sepsis, also appears to be a potent stimulant of thrombogenesis, and warrants attention as another potential mediator of AngII-enhanced microvascular thrombosis.

Oxidative stress and the enhanced production of superoxide, which accompanies chronic AngII administration, is another potent pro-coagulant/prothrombotic stimulant^{8, 14}. Reactive oxygen species, including superoxide and secondarily-derived oxidants, are known to augment platelet aggregation responses, activate the coagulation cascade, inhibit anticoagulant mechanisms and fibrinolysis, and promote thrombus formation^{38, 39}. The principal molecular target of AngII that explains its pro-oxidative effect is NAD(P)H oxidase^{8, 12}. Chronic AngII administration results in an increased expression Nox1, gp91^phox, p47^phox, and p22^phox subunits of NADPH oxidase and leads to increased enzyme activity^{5, 40}. The AngII-mediated enzyme activation is evident in endothelial cells, vascular smooth muscle, and different blood cell populations, including T-lymphocytes^{12, 41, 42}. While vessel wall-associated NADPH oxidase has frequently been linked to the vasomotor and inflammatory effects of AngII, recent evidence supports a role for T-lymphocyte-associated NAD(P)H oxidase in mediating these responses to AngII⁵.

A novel finding of the present study is that NADPH oxidase also contributes to the prothrombotic actions of AngII, as evidenced by our observation that gp91^phox−/− mice do not exhibit the accelerated thrombosis response to AngII that is observed in WT mice. We also assessed the possibility that NADPH oxidase associated with T-cells is responsible for the AngII mediated thrombosis. Our finding that Rag-1^−/− mice reconstituted with T-cells derived from either WT or gp91^phox−/− mice exhibited a fully restored thrombosis response to AngII suggests that NADPH oxidase in T-cells is not critical for AngII-induced thrombosis. While the cellular source of NADPH oxidase that mediates AngII-enhanced thrombosis response remains unclear, vascular endothelium or a blood cell population other than T-cells (e.g., monocytes, platelets) are likely candidates. Irrespective of the cellular source of NADPH oxidase that contributes to AngII-enhanced thrombogenesis, our observation that T-lymphocyte deficiency (Rag-1^−/− and CD4⁺ T-cell^−/− mice) and NADPH oxidase deficiency (gp91^phox−/− mice) are equally effective in preventing the AngII mediated acceleration of thrombosis suggests that T-cells and NADPH oxidase are series coupled effectors of the thrombogenic response. One possibility is that AngII activates vascular wall NADPH oxidase, which in turn leads to activation of T-cells, perhaps through the generation of neoantigens secondary to oxidative protein modification^{43, 44}. Alternatively, AngII may activate T-cells, which in turn release mediators, such as cytokines, that increase the expression and activity of NADPH oxidase in endothelium or other blood cell populations. The latter possibility appears more likely since it has been reported that Rag-1^−/− mice do not exhibit the increased vascular superoxide production observed in wild type mice during chronic AngII infusion⁵. Further support is provided by reports describing the ability of T-cell derived cytokines, such as IL-6 and TNF-α, to increase the expression of NADPH oxidase in vascular endothelium^{45, 46}.

Perspectives.

Angiotensin II exerts a prothrombotic effect in both large arteries and arterioles. Immune cell activation and oxidative stress have been implicated in the altered vascular reactivity and accelerated atherothrombosis that accompanies angiotensin II-induced hypertension. The findings of this study are consistent with a role for activation of the adaptive immune system in the accelerated microvascular thrombosis that results from chronically elevated levels of angiotensin II. CD4⁺ T-lymphocytes appear to make a major contribution in this regard. Reactive oxygen species generated from NADPH oxidase is also linked to the prothrombotic action of angiotensin II in arterioles. These observations offer novel therapeutic targets for the prevention of thrombosis in patients with hypertension and/or other risk factors for cardiovascular disease.