Benefits and Risks of Aliskiren Treatment in Patients With Type 2 Diabetes: Analyses of the 3A Registry.

The authors sought to retrospectively analyze the real-world evidence on aliskiren in diabetic patients with or without concomitant renin-angiotensin system (RAS) blocker use based on the Registry for Ambulant Therapy With RAS Inhibitors in Hypertension Patients in Germany (3A). Of 14,986 patients included, 3772 patients had diabetes and 28.5% received aliskiren, 14.3% received angiotensin-converting enzyme (ACE) inhibitors/angiotensin receptor blockers (ARBs), 35.4% received aliskiren plus an ACE inhibitor/ARB, and 10.5% received other drugs. Ambulatory blood pressure (BP) monitoring (baseline BP 148±15.8/84.0±10.9 mm Hg) revealed stronger diastolic BP reduction for aliskiren plus ACE inhibitor/ARB than aliskiren alone in the low (2.8±0.5 vs 0.6±0.6; P=.004) and intermediate (5.9±0.5 vs 4.5±0.5; P=.04) baseline BP groups. There was a lesser ambulatory BP reduction observed for patients receiving non-RAS in the high baseline category for both systolic (12.5±1.8 vs 17.1±1.0; P=.02) and diastolic (6.9±1.0 vs 9.8±0.6; P=.01) BP. In patients with hypertension and type 2 diabetes, aliskiren was beneficial in lowering BP, with no observed increases in major adverse effects compared with RAS-blocking therapy alone.

Platelet Gi protein Gαi2 is an essential mediator of thrombo-inflammatory organ damage in mice.

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The Gi protein Gα(i2) mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα(i2) exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα(i2) in megakaryocytes and platelets (Gna(i2)(fl/fl)/PF4-Cre mice) and found bleeding defects comparable to those in global Gα(i2)-deficient mice. To examine the impact of platelet Gα(i2) in postischemic thrombo-inflammatory infarct progression, Gna(i2)(fl/fl)/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gna(i2)(fl/fl)/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gna(i2)(fl/fl)/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα(i2) not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.

Disproportional decrease in office blood pressure compared with 24-hour ambulatory blood pressure with antihypertensive treatment: dependency on pretreatment blood pressure levels.

The long-term relationship between 24-hour ambulatory blood pressure (ABP) and office BP in patients on therapy is not well documented. From a registry we included all patients in whom antihypertensive therapy needed to be uptitrated. Drug treatment included the direct renin inhibitor aliskiren or an angiotensin-converting enzyme inhibitor/angiotensin receptor blocker or drugs not blocking the renin-angiotensin system, alone or on top of an existing drug regimen. In all patients, office BP and 24-hour ABP were obtained at baseline and after 1 year with validated devices. In the study population of 2722 patients, there was a good correlation between the change in office BP and 24-hour ABP (systolic: r=0.39; P<0.001; diastolic: r=0.34; P<0.001). However, the numeric decrease in office BP did not correspond to the decrease in ABP in a 1:1 fashion, for example, a decrease of 10, 20, and 30 mm Hg corresponded to a decrease of ≈7.2, 10.5, and 13.9 mm Hg in systolic ABP, respectively. The disproportionally greater decrease in systolic office BP compared with ABP was dependent on the level of the pretreatment BP, which was consistently higher for office BP than ABP. The white coat effect (difference between office BP and ABP) was on average 10/5 mm Hg lower 1 year after intensifying treatment and the magnitude of that was also dependent on pretreatment BP. There was a disproportionally greater decrease in systolic office BP than in ABP, which for both office BP and ABP seemed to depend on the pretreatment BP level.

Combined in vivo depletion of glycoprotein VI and C-type lectin-like receptor 2 severely compromises hemostasis and abrogates arterial thrombosis in mice.

OBJECTIVE: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. APPROACH AND RESULTS: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. CONCLUSIONS: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.

Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction.

Myocardial infarction (MI) leads to rapid necrosis of cardiac myocytes. To achieve tissue integrity and function, inflammatory cells are activated, including monocytes/macrophages. However, the effect of monocyte/macrophage recruitment after MI remains poorly defined. After experimental MI, monocytes and macrophages were depleted through serial injections of clodronate-containing liposomes. Monocyte/macrophage infiltration was reduced in the myocardium after MI by active treatment. Mortality was increased due to thromboembolic events in monocyte- and macrophage-depleted animals (92 vs. 33%; P<0.01). Left ventricular thrombi were detectable as early as 24 h after MI; this was reproduced in a genetic model of monocyte/macrophage ablation. A general prothrombotic state, increased infarct expansion, and deficient neovascularization were not observed. Severely compromised extracellular matrix remodeling (collagen I, placebo liposome vs. clodronate liposome, 2.4 ± 0.2 vs. 0.8 ± 0.2 arbitrary units; P<0.001) and locally lost integrity of the endocardium after MI are potential mechanisms. Patients with a left ventricular thrombus had a relative decrease of CD14CD16 monocyte/macrophage subsets in the peripheral blood after MI (no thrombus vs. thrombus, 14.2 ± 0.9 vs. 7.80 ± 0.4%; P<0.05). In summary, monocytes/macrophages are of central importance for healing after MI. Impaired monocyte/macrophage function appears to be an unrecognized new pathophysiological mechanism for left ventricular thrombus development after MI.

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature.

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

Altered microtubule equilibrium and impaired thrombus stability in mice lacking RanBP10.

The crucial function of blood platelets in hemostasis is to prevent blood loss by stable thrombus formation. This process is driven by orchestrated mechanisms including several signal transduction cascades and morphologic transformations. The cytoplasmic microtubule modulator RanBP10 is a Ran and ß1-tubulin binding protein that is essential for platelet granule release and mice lacking RanBP10 harbor a severe bleeding phenotype. In this study, we demonstrate that RanBP10-nullizygous platelets show normal adhesion on collagen and von Willebrand factor under flow conditions. However, using a ferric chloride-induced arterial thrombosis model, the formation of stable thrombi was significantly impaired, preventing vessel occlusion or leading to recanalization and thromboembolization. Delta-granule secretion was normal in mutant mice, whereas platelet shape change in aggregometry was attenuated. Lack of RanBP10 leads to increased ß1-tubulin protein, which drives α-monomers into polymerized microtubules. In mutant platelets agonists failed to contract the peripheral marginal band or centralize granules. Pretreatment of wild-type platelets with taxol caused microtubule stabilization and phenocopied the attenuated shape change in response to collagen, suggesting that RanBP10 inhibits premature microtubule polymerization of ß1-tubulin and plays a pivotal role in thrombus stabilization.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis.

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

Early detrimental T-cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor thrombus formation.

T cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T-cell receptors (TCRs) and mutations in costimulatory molecules were used to define the minimal immunologic requirements for T cell-mediated ischemic brain damage. Stroke was induced in recombination activating gene 1-deficient (RAG1(-/-)) mice devoid of T and B cells, RAG1(-/-) mice reconstituted with B cells or T cells, TCR-transgenic mice bearing 1 single CD8(+) (2C/RAG2, OTI/RAG1 mice) or CD4(+) (OTII/RAG1, 2D2/RAG1 mice) TCR, mice lacking accessory molecules of TCR stimulation (CD28(-/-), PD1(-/-), B7-H1(-/-) mice), or mice deficient in nonclassical T cells (natural killer T [NKT] and gammadelta T cells) by transient middle cerebral artery occlusion (tMCAO). Stroke outcome was assessed at day 1. RAG1(-/-) mice and RAG1(-/-) mice reconstituted with B cells developed significantly smaller brain infarctions compared with controls, but thrombus formation after FeCl(3)-induced vessel injury was unimpaired. In contrast, TCR-transgenic mice and mice lacking costimulatory TCR signals were fully susceptible to tMCAO similar to mice lacking NKT and gammadelta T cells. These findings were corroborated by adoptive transfer experiments. Our data demonstrate that T cells critically contribute to cerebral ischemia, but their detrimental effect neither depends on antigen recognition nor TCR costimulation or thrombus formation.

Factor XIIa inhibitor recombinant human albumin Infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding.

BACKGROUND: Blood coagulation is a tightly regulated process of sequentially activated serine proteases culminating in fibrin formation, which is critical for limiting posttraumatic blood loss but also may contribute to acute thrombotic diseases, most notably myocardial infarction and stroke. Recent studies with factor XII-deficient mice revealed that the factor XII-induced intrinsic coagulation pathway is essential for pathological thrombus formation but dispensable for hemostasis. Consequently, these findings led to the hypothesis that factor XII could be a promising pharmacological target for safe antithrombotic therapy. METHODS AND RESULTS: The complementary DNA of the previously described factor XIIa inhibitor Infestin-4, cloned from the midgut of Triatoma infestans, was fused to recombinant human albumin (rHA) and analyzed in vitro. The resulting protein rHA-Infestin-4 specifically inhibits factor XIIa and causes prolonged activated partial thromboplastin time in human, mouse, and rat plasma. To assess its inhibitory potency in vivo, mice and rats were injected with rHA-Infestin-4 and challenged in pathological thrombus formation models. In addition, bleeding assays were performed. rHA-Infestin-4 completely abolished occlusive arterial thrombus formation in mice and rats while leaving hemostasis fully intact. Furthermore, rHA-Infestin-4 was highly protective in a murine model of ischemic stroke. CONCLUSIONS: These results identify rHA-Infestin-4 as a promising agent to achieve powerful protection from ischemic cardiovascular and cerebrovascular events without affecting hemostasis.

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets.

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, alpha(IIb)beta(3) integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate-binding protein or immunoreceptor tyrosine-based activation motif-coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1(-/-) mice displayed impaired alpha(IIb)beta(3) integrin activation in response to major agonists and defective glycoprotein Ib-dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis.

Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti-CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2-deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.