Rac1 Modulates Endothelial Function and Platelet Aggregation in Diabetes Mellitus.

BACKGROUND: Vascular complications and abnormal platelet function contribute to morbidity and mortality in diabetes mellitus. We hypothesized that the Rho-related GTPase protein, Rac1, can influence both endothelial and platelet function and might represent a potential novel therapeutic target in diabetes mellitus. METHODS AND
RESULTS: We used both in vitro and ex vivo approaches to test the effects of pharmacological inhibition of Rac1 during hyperglycemic condition. We evaluated the effect of NSC23766, a pharmacological inhibitor of Rac1, on vascular function in diabetic mice and platelet aggregation in diabetic subjects. We demonstrated that the administration of NSC23766 protects from hyperglycemia-induced endothelial dysfunction, restoring NO levels, and reduces oxidative stress generated by nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, we identified Rho-associated coiled-coil serine/threonine kinase-1 as a downstream target of Rac1. Moreover, we reported that during hyperglycemic conditions, human platelets showed hyperactivation of Rac1 and impaired NO release, which were both partially restored after NSC23766 treatment. Finally, we characterized the antiplatelet effect of NSC23766 during hyperglycemic conditions, demonstrating the additional role of Rac1 inhibition in reducing platelet aggregation in diabetic patients treated with common antiplatelet drugs.
CONCLUSIONS: Our data suggest that the pharmacological inhibition of Rac1 could represent a novel therapeutic strategy to reduce endothelial dysfunction and platelet hyperaggregation in diabetes mellitus.

Clinical Perspective

What Is New?

The molecular mechanisms that govern endothelial dysfunction and enhanced platelet aggregation in diabetes mellitus are not completely elucidated.

Herein, we show that Rac1 participates in diabetes mellitus–induced platelet alterations and endothelial dysfunction.

What Are the Clinical Implications?

Rac1 inhibition reduces platelet hyperactivity and endothelial dysfunction in diabetes mellitus.

Therefore, Rac‐1 could represent a potential therapeutic target to ameliorate both pathophysiological alterations in diabetes mellitus.

Introduction

Macrovascular and microvascular complications contribute to morbidity and mortality in diabetes mellitus.1, 2, 3 Endothelial dysfunction and abnormal platelet function represent the main determinants of the vascular accidents in diabetic patients, contributing to high incidence of thrombotic events.4 Chronic hyperglycemia observed in type 2 diabetes mellitus induces platelet activation and increases reactive oxygen species (ROS) production in endothelium, playing an important role in the development of vascular damage.5, 6

The small GTPase Rac1 is essential for the correct assembly of nicotinamide adenine dinucleotide phosphate oxidase (Nox) subunits.7 Several pathways converge in the activation of Rac1, and some evidence suggests a role in different cellular mechanisms, such as cell adhesion, chemotaxis, and vascular permeability.7 In addition to its role in ROS generation in endothelium, Rac1 represents a key orchestrator of platelet actin cytoskeleton, modulating, in turn, platelet aggregation.8 The role of Rac1 in hyperglycemia‐induced platelet hyperaggregation is still poorly understood.

Antiplatelet drugs, such as aspirin, are prescribed to diabetic patients for prevention of ischemic cardiovascular diseases; however, many patients exhibit “aspirin resistance” with a high rate of cardiovascular events.9, 10, 11 Furthermore, despite optimal antiplatelet therapy, many patients exhibit high residual platelet reactivity, which has been associated with higher risk of cardiovascular events as well.12 Therefore, even a small variation in platelet activity might precipitate thrombotic events. This indicates the necessity of identifying new molecular targets to limit platelet aggregation in diabetes mellitus. In this regard, Rac1 could represent a good candidate able to modulate both endothelial function and platelet aggregation. Recently, a small molecule able to inhibit Rac1 activity, named NSC23766, has been developed.13, 14 NSC23766 has been shown to inhibit Rac1 activity by interfering with its binding domain involved in the determination of Rac1's specificity to a subset of guanine nucleotide exchange factors that catalyze the exchange of GDP to GTP to maintain Rac1 in its active, GTP‐bound, form.14, 15 Recently, we have shown the beneficial effects of Rac1 inhibition through NSC23766 on endothelial dysfunction in human vessels.16 However, the mechanisms by which NSC23766 exerts its protective role on endothelial function in hyperglycemia are still partially unknown.

Rho‐associated coiled‐coil serine/threonine kinase‐1 (ROCK1) is the main downstream target of the small GTPase RhoA and has been involved in the regulation of multiple cellular functions involving cytoskeletal organization.17 Given the important role of Rac1 in endothelial and platelet function, we hypothesized that ROCK1 might represent a potential downstream target of Rac1 activity.

To better understand the role of Rac1 in diabetes mellitus, we investigated the possible therapeutic role of NSC23766 on vascular and platelet alteration using both in vitro and ex vivo approaches in preclinical model of diabetes mellitus and human samples.

Methods

The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure on request to corresponding authors. All experiments involving animals were conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication 85‐23, revised 2011) and were approved by the Istituto di Ricovero e Cura a Carattere Scientifico Istituto Neurologico Mediterraneo Neuromed review board. Human subjects were enrolled at the Cardiology Division of the University of Naples Federico II. The study protocol was conformed to the principles outlined in the Declaration of Helsinki and was approved by the institutional review board of the medical center, and each patient who accepted to participate provided written informed consent. All diabetic patients enrolled in our study fulfilled the criteria of the National Diabetes Data Group for diabetes mellitus.18 Characteristics of patients (demographics, concomitant medication therapy, and glycemic status) are summarized in the Table and Table S1. An expanded description of materials and methods used in this study is available in Data S1.

Table 1

Baseline Characteristics of the Study Subjects

Characteristics	Control Subjects (n=11)	Diabetic Subjects (n=22)
Age, y	56.3±5.03	57.05±2.39
Women, %	36	30
Hypertension, %	40	35
Hyperlipidemia, %	22	25
BMI, kg/m2	24.17±1.1	26.13±1.12
Smoking, %	46	45
HbA1c, %	5.5±0.31	8.3±1.32a
Drug therapy, n
Statins	0	0
ACEIs	6	10
ARBs	6	10
CCBs	5	10
BBs	4	5
Anticoagulants	0	0
ASA	0	7
Other antiplatelet agents	0	0

Data are represented as mean±SD unless otherwise indicated. ACEI indicates angiotensin‐converting enzyme inhibitor; ARB, angiotensin receptor blocker; ASA, acetylsalicylic acid; BB, β blocker; BMI, body mass index; CCB, calcium channel blocker; and HbA1c, glycated hemoglobin.

P=0.00012 vs control subjects.

Statistical Analysis

Data are presented as bar graphs, box‐and‐whisker plots, or points and connecting line. Plots show mean, and the error bars represent SEM. Different data sets were generated to test differences in the experiments involving animals/cells or humans. In the animal/cells study, differences were analyzed by Mann‐Whitney nonparametric test to compare 2 independent groups or by Kruskal‐Wallis test in experiments including ≥3 groups. For vascular reactivity studies, differences were analyzed using nonparametric Friedman test, followed by Dunn's multiple comparison test, for the analysis of the effects of the pharmacological treatments on vascular reactivity function. Human data were presented as mean and SD and analyzed by 2‐tailed Student t test (Table). No randomization was applied to allocate patients in the different groups because diabetic patients were chosen on the basis of glycated hemoglobin percentage (baseline difference). Therefore, no adjustment in the analysis was made because of the baseline differences. No repeated measurements on the same experimental unit over time were used. All experiments can be considered on different experimental units. A minimum value of P<0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software 7.0.

Results

Rac1 Inhibition Protects From Endothelial Dysfunction in a Mouse Model of Diabetes Mellitus

To evaluate the effects of NSC23766 on vascular function in diabetes mellitus, we used a previously described mouse model of streptozotocin‐induced diabetes mellitus.19 Mesenteric arteries were isolated from streptozotocin‐treated mice and their control (vehicle‐injected) littermates after IP injection of NSC23766 (5 mg/kg, as previously described),20, 21 at different time points (6, 12, 24, 36, 48, and 96 hours after injection) to perform vascular reactivity studies (Figure 1A through 1F). No effects on blood glucose levels and body weight were found after NSC23766 treatment in both control and streptozotocin‐treated mice (Table S2).

Figure 1

NSC23766 restores relaxation in diabetic vessels. Acetylcholine (ACh) vasorelaxation in preconstricted mesenteric arteries from vehicle‐treated mice (control [Ctrl]; full circles), streptozotocin‐treated mice (STZ; empty circles), from Ctrl mice treated with Rac1 inhibitor (Ctrl+NSC23766; full squares), and from STZ‐treated mice plus Rac1 inhibitor (STZ+NSC23766; empty squares) at different time points from single injection of NSC23766: 6 hours (A), 12 hours (B), 24 hours (C), 36 hours (D), 48 hours (E), and 96 hours (F). n=4 for each group. *P<0.05 vs all.

As expected, diabetes mellitus caused impaired endothelial vasorelaxation, as demonstrated by reduced response to acetylcholine in mesenteric arteries of mice treated with streptozotocin (Figure 1). In contrast, smooth muscle relaxation induced by nitroglycerine was unaffected by diabetes mellitus (data not shown). Interestingly, in vivo administration of NSC23766 in streptozotocin‐treated mice reduced endothelial dysfunction, ameliorating vasorelaxation starting after 6 hours from injection, with a sustained effect present up to 96 hours after administration (Figure 1A through 1F).

As expected, diabetic arteries also exhibited increased ROS production and Nox activity (Figure 2A). We also observed that, in streptozotocin‐treated mice, both mRNA and protein levels of ROCK1 were increased (Figure 2B and 2C), coupled with significant downregulation of phosphoinositide 3‐kinase/protein kinase B signaling pathway (Figure 2C). Interestingly, NSC23766 treatment abolished Rac1 activation in diabetic vessels, which, in turn, reduced RhoA and ROCK1 levels, restoring the phosphoinositide 3‐kinase/protein kinase B signaling pathway and endothelial NO synthase (eNOS) phosphorylation (Figure 2B). These data support the role of Rac1 as an upstream modulator of ROCK1 involved in eNOS dysfunction and ROS production in diabetes mellitus.

Figure 2

NSC23766 restores endothelial NO synthase (eNOS) function and reduces reactive oxygen species in diabetic vessels. A, Representative micrographs of Dihydroethidium staining to evaluate oxidative stress in mesenteric arteries from mice treated with vehicle (control [Ctrl]), with streptozotocin (STZ), or with streptozotocin plus NSC23766 (STZ+NSC23766; 48 hours). Representative images (n=3). Columns represent the effect of NSC23766 on nicotinamide adenine dinucleotide phosphate (NADPH)–induced lucigenin chemiluminescence in STZ mice mesenteric arteries. Data are expressed as increase of chemiluminescence per minute in arbitrary units. n=4 for each group. *P<0.05 vs all. B, The mRNA levels of Rho‐associated coiled‐coil serine/threonine kinase‐1 (ROCK1) were determined by quantitative reverse transcription–polymerase chain reaction in vessels from Ctrl, STZ, and STZ+NSC23766, 48 hours. n=3 for each group. *P<0.05. C, Representative immunoblots (left) and densitometric analysis (right) of 4 independent experiments evaluating protein levels of ROCK‐1, phospo (p)‐eNOS, eNOS, p‐phosphoinositide 3‐kinase (PI3K), PI3K, p‐protein kinase B (Akt; T473), Akt, p21 activated kinase, RhoA‐GPT, RhoA, Rac1‐GTP, Rac1, and β‐actin in mesenteric arteries from Ctrl, STZ, and STZ+NSC23766 mice, 48 hours. n=3 for each group. *P<0.05.

NSC23766 Prevents High Glucose–Induced Endothelial Dysfunction by Restoring eNOS Phosphorylation and Reducing Oxidative Stress

To evaluate the in vitro effects of NSC23766 on glucose‐induced endothelial dysfunction, mesenteric arteries from wild‐type C57BL/6 mice were treated with 2 different glucose concentrations, mimicking normoglycemia (5 mmol/L) or hyperglycemia (25 mmol/L). Vessels exposed to 25 mmol/L of glucose for 30 minutes showed a significant reduction of acetylcholine‐evoked vasorelaxation compared with vessels treated with 5 mmol/L of glucose (Figure 3A), whereas no differences between the 2 different doses of glucose were found in nitroglycerine‐induced vasorelaxation (Figure S1). These data confirm the detrimental effects of high glucose levels on vascular function. Interestingly, pretreatment with Rac1 inhibitor, NSC23766 (30 μmol/L), was able to protect from endothelial dysfunction induced by high glucose (Figure 3A), restoring eNOS phosphorylation and reducing ROS production and Nox activity (Figure 3B and 3C).

Figure 3

Interplay between RAC1 and Rho‐associated coiled‐coil serine/threonine kinase‐1 in vessels and platelets. A, Acetylcholine (ACh) vasorelaxation in preconstricted mesenteric arteries treated with low glucose (Glu; 5 mmol/L; full circles), high glucose (Glu 25 mmol/L; empty circles), low glucose plus Rac1 inhibitor (Glu 5 mmol/L+NSC23766; full squares), and high glucose plus Rac1 inhibitor (Glu 25 mmol/L+NSC23766; empty squares). n=4 for each group. *P<0.05 vs all. B, Representative immunoblots (left) and densitometric analysis (right) of 4 independent experiments evaluating protein levels of p‐endothelial NO synthase (eNOS), eNOS, Rac1‐GTP, total Rac1, and β‐actin in mesenteric arteries treated with Glu 5 mmol/L, Glu 25 mmol/L, Glu 25 mmol/L plus LY27632 (Glu 25 mmol/L+LY27632), Glu 25 mmol/L+NSC23766, or Glu 25 mmol/L+NSC23766+LY27632. *P<0.05 vs all; *P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+NSC23766, and Glu 25 mmol/L+NSC23766+LY27632. C, Representative images of Dihydroethidium staining to evaluate oxidative stress in mesenteric arteries treated with different stimuli/inhibitors. Columns represent the effect of NSC23766, LY27632, GKT137831, and ML‐171 on nicotinamide adenine dinucleotide phosphate (NADPH)–induced lucigenin chemiluminescence in mice mesenteric arteries. Data are expressed as increase of chemiluminescence per minute in arbitrary units. n=4 for each group. *P<0.05. D, ACh vasorelaxation in preconstricted mesenteric arteries from control treated with Glu 5 mmol/L, Glu 25 mmol/L, Glu 25 mmol/L+LY27632, Glu 25 mmol/L+NSC23766, and Glu 25 mmol/L+NSC23766+LY27632. n=4 for each group. *P<0.05 vs Glu+LY27632; # P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+NSC23766, and Glu 25 mmol/L+NSC23766+LY27632; § P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+NSC23766, and Glu 25 mmol/L+NSC23766+LY27632. E, ACh vasorelaxation in preconstricted mesenteric arteries from mesenteric arteries treated with Glu 5 mmol/L, Glu 25 mmol/L, Glu 25 mmol/L+LY27632, Glu 25 mmol/L+LY27632+tiron, Glu 25 mmol/L+NSC23766+tiron, and Glu 25 mmol/L+tiron. n=3 for each group. *P<0.05 vs Glu 25 mmol/L+LY27632; # P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+LY27632+tiron, and Glu 25 mmol/L+NSC23766+tiron; § P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+LY27632+tiron, and Glu 25 mmol/L+NSC23766+tiron; ° P<0.05 vs Glu 5 mmol/L, Glu 25 mmol/L+LY27632+tiron, and Glu 25 mmol/L+NSC23766+tiron. F, ACh vasorelaxation in preconstricted mesenteric arteries from vessels treated with Glu 25 mmol/L, Glu 25 mmol/L plus ML‐171 (Glu 25 mmol/L+ML‐171), or Glu 25 mmol/L plus GTK137831 (Glu 25 mmol/L+GTK137831. n=5 for each group. *P<0.05 vs Glu25 mmol/L+GTK137831; # P<0.05 vs Glu 25 mmol/L+ML‐171.

ROCK1 Is Involved in Rac1‐Dependent Effects on Vascular Function

It has been reported that Rac1 negatively modulates eNOS function.22 This mechanism appears to be likely mediated by the reduction of ROCK1, a negative regulator of eNOS. In fact, ROCK1 inhibits eNOS gene expression and inhibits phosphoinositide 3‐kinase/protein kinase B signaling, which phosphorylates and activates eNOS.23, 24 Accordingly, vessels treated with high glucose concentration in presence of LY27632, an inhibitor of ROCK1 activity, showed increased eNOS phosphorylation and enhanced vasorelaxation compared with vessels treated with high glucose alone (Figure 3B and 3D). Notably, in LY27632‐treated vessels, Rac1 was still activated, positioning it as an upstream modulator of ROCK1 (Figure 3B). Although the administration of Rac1 inhibitor in presence of LY27632 did not further enhance eNOS phosphorylation compared with LY27632 alone (Figure 3B), at functional level it was able to potentiate endothelial vasorelaxation, suggesting that additional mechanism(s) are recruited by Rac1 inhibitor to modulate endothelial function (Figure 3D). Interestingly, in the same model of hyperglycemia‐induced vascular damage, inhibition of Rac1 but not inhibition of ROCK1 was able to protect the arteries from high glucose–induced ROS generation, as shown by a significant reduction in Nox levels after treatment with NSC23766 (Figure 3C).

To better evaluate the contribution of ROS in the differential response on vascular function observed with Rac1 and ROCK1 inhibitors, the antioxidant agent tiron (10−3 mol/L), a superoxide scavenger, was used in addition to ROCK1 inhibitor. Treatment with tiron+LY27632 restored endothelial vasorelaxation at similar level observed in presence of Rac1 inhibitor (Figure 3E). On the contrary, the addition of tiron to NSC23766 did not influence the vascular response evoked by Rac1 inhibition in presence of high glucose levels, whereas treatment with tiron alone only partly ameliorated vascular relaxation, accordingly with its scavenger effects (Figure 3E).

Given the striking reduction of ROS production and Nox activity observed after NSC23766 treatment, we aimed to determine the individual contribution of different Nox isoforms in glucose‐induced ROS generation. Interestingly, the use of Nox4 inhibitor, GTK137831, significantly decreased ROS production and Nox activity in glucose‐treated vessels (Figure 3C), whereas no effects were observed after treatment with Nox1 inhibitor, ML‐171 (Figure 3C). We also observed that Nox4 inhibition by GTK137831 partially restored acetylcholine‐induced vasorelaxation in mesenteric arteries treated with high glucose dose (Figure 3F). Interestingly, treatment of dysfunctional vessels with Nox1 inhibitor ML‐171 did not improve vascular reactivity (Figure 3F). These data demonstrate a specific involvement of Nox4 isoform in glucose‐induced ROS production and suggest that the observed vascular antioxidant effects of NSC23766 could be mediated, at least in part, by the inhibition of Nox4 isoform of Nox.

The beneficial effects of Rac1/ROCK1 inhibition on glucose‐induced endothelial dysfunction and ROS production observed in the whole vessels were also observed in human endothelial cells (Figure S2). Collectively, these data demonstrate that ROCK1 inhibition ameliorates, in part, hyperglycemia‐induced endothelial dysfunction without affecting ROS production, whereas the improvement of endothelial function observed with Rac1 inhibitor is also attributable to its inhibitor effect on Nox, primarily on Nox4 isoform, reducing ROS production.

Rac1 Inhibition Restores NO Production in High Glucose–Treated Human Platelets

Treatment of human platelets with high glucose concentration (25 mmol/L) induced a strong activation of Rac1 and a significant reduction of eNOS phosphorylation (Figure 4A). Interestingly, administration of LY27632 restored eNOS phosphorylation without affecting Rac1 activation (Figure 4A), posing Rac1 as an upstream modulator of ROCK1 signaling also in platelets. In addition, NSC23766 was also able to restore eNOS phosphorylation, in presence of high glucose, at similar levels compared with LY27632, whereas coadministration of both Rac1 and ROCK1 inhibitors did not further modify eNOS phosphorylation status (Figure 4A). These data suggested that the effect of NSC23766 on eNOS phosphorylation is mediated by ROCK1.

Figure 4

Rac1 inhibition restores NO release from platelets. A, Representative immunoblots (left) and densitometric analysis (right) of 4 independent experiments evaluating protein levels of p‐endothelial NO synthase (eNOS), eNOS, Rac1‐GTP, total Rac1, and β‐actin in platelets. *P<0.05 vs all. B, Quantitative measurement of NO levels in platelet supernatants treated with glucose 5 mmol/L (Glu 5 mmol/L), Glu 5 mmol/L+Nω‐nitro‐l‐arginine methyl ester hydrochloride (L‐NAME), glucose 25 mmol/L (Glu 25 mmol/L), Glu 25 mmol/L plus NSC23766 (Glu 25 mmol/L+NSC23766), Glu 25 mmol/L plus LY27632 (Glu 25 mmol/L+LY27632), Glu 25 mmol/L plus NSC23766 plus LY27632 (Glu 25 mmol/L+NSC23766+LY27632), and Glu 25 mmol/L plus LY27632 plus tiron (Glu 25 mmol/L+LY27632+tiron). Box plots representing the mean and the minimum and maximum values of NO amounts. n=4 for each group. *P<0.05 vs all; # P<0.05 vs Glu 25 mmol/L+NSC23766, Glu 25 mmol/L+NSC23766+LY27632, Glu 5 mmol/L, and Glu 25 mmol/L. C, Dose‐response curves of phenylephrine precontracted aorta rings to supernatants derived from human platelets treated with Glu 5 mmol/L, Glu 5 mmol/L+L‐NAME, and Glu 25 mmol/L. n=4 for each group. *P<0.05 vs Glu 5 mmol/L. D, Dose‐response curves of phenylephrine precontracted aorta rings to supernatants derived from human platelets treated with Glu 5 mmol/L, Glu 25 mmol/L, Glu 25 mmol/L+NSC23766, Glu 25 mmol/L+LY27632, Glu 25 mmol/L+NSC23766+LY27632, Glu 25 mmol/L+LY27632+tiron, or Glu 25 mmol/L+tiron. n=4 for each group. *P<0.05 vs all; § P<0.05 vs Glu 25 mmol/L+LY27632; § P<0.05 vs Glu 25 mmol/L+NSC23766; # P<0.05 vs all.

Reduced platelet NO production represents a crucial alteration in diabetes mellitus. As expected, we observed a dramatic impairment of NO release in the supernatants of platelets exposed to high glucose concentration compared with platelets exposed to low glucose, measured by Sievers NO analyzer (NOA280i) (Figure 4B). To confirm the specificity of NO production, we also measured NO levels in supernatant of platelets treated with NOS inhibitor Nω‐nitro‐l‐arginine methyl ester hydrochloride. As shown in Figure 4B, Nω‐nitro‐l‐arginine methyl ester hydrochloride treatment completely abolished NO production in platelet supernatant. Interestingly, treatment with Rac1 inhibitor NSC23766 restored platelet NO production, whereas treatment of high glucose–stimulated platelets with ROCK1 inhibitor was able to restore only, in part, the impaired NO production (Figure 4B). Similar to what was observed in vessels, addition of tiron to high‐glucose platelets treated with LY27632 further enhanced NO production to the levels observed with Rac1 inhibitor NSC23766 (Figure 4B).

Human platelet supernatant evoked a rapid dose‐dependent relaxation of mouse aortic rings; this effect was NO dependent because it was abolished by eNOS inhibition with Nω‐nitro‐l‐arginine methyl ester hydrochloride (Figure 4C). As expected, the effect on vasorelaxation induced by platelet supernatant was markedly reduced by high glucose level (25 mmol/L) compared with vessels treated with supernatant of platelets with low dose of glucose (5 mmol/L) (Figure 4C), confirming the effects of high glucose to induce an impairment in the NO‐dependent vasorelaxant mechanisms.

To evaluate the role of Rac1/ROCK1 axis in platelet‐induced NO‐dependent vasorelaxation, supernatants from NSC23766 or ROCK1 inhibitor LY27632‐treated platelets were used on mouse aortic ring preparations. Interestingly, treatment of platelets with NSC23766 restored the ability of platelet supernatant to induce a dose‐dependent relaxation of mouse aortic rings in presence of high glucose levels (Figure 4D), whereas the supernatant of LY27632‐treated platelets was able to ameliorate only, in part, vasorelaxation (Figure 4D). Interestingly, the addition of tiron caused an enhancement of vasorelaxant effect observed with supernatant of LY27632‐treated platelets, reaching a similar level on what was observed in presence of NSC23766 alone or NSC23766 plus LY27632 (Figure 4D). The administration of tiron alone exerted only a mild, not significant, improvement of vasorelaxation.

Taken together, these results indicated that the effect of Rac1 inhibitor on NO metabolism in presence of high glucose levels depends on the modulation of both eNOS phosphorylation and oxidative stress.

NSC23766 Reduces Platelet Aggregation Induced by High Glucose Levels

Because impairment of NO production is closely associated with an increase of platelet reactivity, we investigated the effect of Rac1 inhibitor on human platelet aggregation. As expected, platelet aggregation induced by type I collagen was enhanced after treatment with increasing concentrations of glucose (Figure 5A). Subsequently, we aimed to identify the effective concentration of NSC23766 able to modulate platelet aggregation. We performed a dose‐response curve with NSC23766 in human platelets exposed to 5 and 25 mmol/L of glucose. In platelets exposed to 5 mmol/L (mimicking “normoglycemic” condition), the effective dose to reach a significant inhibition of platelet aggregation was 30 μmol/L, whereas further increase in NSC23766 dose (starting from 50 μmol/L) practically abolished any collagen‐induced aggregation (Figure 5B). Interestingly, when the same dose‐response curve was done exposing platelets to 25 mmol/L of glucose (mimicking “hyperglycemic” condition), an increased sensitivity to Rac1 inhibition was observed. Specifically, under this condition, the inhibitory effect of NSC23766 on platelet aggregation appeared already at the dose of 15 μmol/L, decreasing further at 30 μmol/L (Figure 5B). Similar results were obtained when platelets were stimulated with different agonists, such as arachidonic acid (0.5 mmol/L), ADP (50 mmol/L), or thrombin receptor‐activating peptide (25 μmol/L) (data not shown). These results suggest that glucose per se can prime platelets, making them more susceptible to the effects of NSC23766, and that Rac1 hyperactivity played a pivotal role in the modulation of high glucose–induced platelet aggregation.

Figure 5

NSC23766 inhibits glucose‐induced platelet hyperaggregation. A, Quantification of platelet aggregation presented as percentage of light transmission of platelets from control (CTRL) subjects treated with increasing concentrations of glucose (Glu). n=6 independent experiments from individual subjects. *P<0.05 vs glucose 5 mmol/L. B, Quantification of platelet aggregation presented as percentage of light transmission of platelets from CTRL subjects treated with increasing concentrations of NSC23766 at 5 and 25 mmol/L Glu. n=4 independent experiments from individual subjects. *P<0.05 vs Glu 5 mmol/L without NSC23766; # P<0.05 vs Glu 5 mmol/L with NSC23766 30 μmol/L; § P<0.05 vs Glu 25 mmol/L without NSC23766; † P<0.05 vs Glu 25 mmol/L with NSC23766 15 μmol/L and Glu 5 mmol/L with NSC23766 30 μmol/L.

Moreover, to demonstrate that Rac1 effects on glucose‐dependent platelet aggregation were not dependent by changes in platelet osmolarity, we evaluated platelet aggregation after increasing dose of osmotic‐control mannitol with and without NSC23766. The administration of mannitol did not change platelet reactivity, confirming the specific role of glucose to induce platelet hyperaggregation (Figure S3). Accordingly, platelet Rac1 levels did not show any change after mannitol treatment (Figure S3).

Finally, we investigated the potential role of ROCK1 in modulation of platelet aggregation during hyperglycemic conditions. Differently from what was observed in vessels, ROCK1 inhibition by LY27632 in platelets did not affect platelet aggregation in presence of both high glucose or low glucose concentrations (data not shown), confirming the marginal role of ROCK1 in modulation of platelet aggregation, as observed before.25

Platelets From Diabetic Patients Show Increased Levels of Activated Rac1

To translate our results into a clinical setting, we evaluated active Rac1 levels in platelets isolated from diabetic patients and control subjects without diabetes mellitus (Table). Platelets from diabetic patients showed higher levels of activated Rac1 compared with control samples (Figure 6A and 6B).

Figure 6

NSC23766 ameliorates platelet hyperaggregation in diabetes mellitus. A, Representative immunoblots of Rac1‐GTP and Rac1 levels in platelets from control (CTRL) subjects or diabetic (Db) patients with different percentage of glycated hemoglobin (HbA1c). GAPDH protein levels were used for normalizing samples. B, Densitometric analysis of Rac1‐GTP (left) and Rac1 (right) protein levels in platelet samples from CTRL and Db patients. n=4 independent experiments from individual subjects. *P<0.05 vs all; # P<0.05 vs CTRL; § P<0.05 vs Db HbA1c 7.0%. C, Quantification of platelet aggregation presented as percentage of light transmission of CTRL platelets (left), Db platelets (middle), and Db platelets+acetylsalicylic acid (ASA) treated with NSC23766. n=4 independent experiments from individual subjects. *P<0.05 vs CTRL 0 (without NSC23766); # P<0.05 vs Db 0 (without NSC23766); § P<0.05 vs CTRL, Db 0, and Db+ASA 0 (without NSC23766). IB indicates immunoblot; NC, negative control; and PC, positive control.

It is well known that in diabetes mellitus, platelets are hyperreactive, with intensified adhesion, activation, and aggregation.26 Thus, we evaluated the level of activated Rac1 in platelets from diabetic patients accordingly with their reported percentage of glycated hemoglobin. Interestingly, our pull‐down assay showed a higher level of active Rac1 in platelets from patients with elevated percentage of glycated hemoglobin (10%) compared with those with lower glycated hemoglobin (7%), indicating a correlation between diabetes mellitus status and Rac1 activation (Figure 6A and 6B).

Moreover, to evaluate the effect of diabetes mellitus on platelets, we evaluated platelet aggregation in diabetic patients. As expected, under basal condition, the aggregation induced by collagen was enhanced in platelets from diabetic patients compared with control subjects (Figure 6C, left panel). Next, we evaluated the efficacy of NSC23766 to inhibit collagen‐induced platelet activation in diabetic patients. Interestingly, to obtain a significant reduction in diabetic platelet aggregation, a higher dose of NSC23766 was necessary compared with control platelets (60 in comparison to 30 μmol/L already effective in platelets from control subjects) (Figure 6C, middle panel).

Although platelets exposed to increasing concentration of glucose exhibit an increased sensitivity to Rac1 inhibition (Figure 5B), higher doses of Rac1 inhibitor were necessary to reach the same levels of inhibition of aggregation in platelets isolated from diabetic patients (Figure 6C, middle panel). To rule out the potential off‐target effects of NSC23766 observed when used at high concentrations,27 we used another structurally different Rac1 inhibitor, called EHT1864. As shown in Figure S4, also with EHT1864, a higher dose of inhibitor was necessary to significantly reduce platelet aggregation in diabetic conditions compared with control subjects.

NSC23766 Exerts Additive Effect on Platelet Aggregation From Diabetic Patients Treated With Acetylsalicylic Acid

On the basis of the evidence that several diabetic patients showed a resistance to common antiplatelet drugs, such as acetylsalicylic acid (ASA), we decided to investigate the efficacy of NSC23766 treatment in isolated platelets from ASA‐treated diabetic patients. In particular, we tested increasing concentrations of NSC23766 on platelets from diabetic patients treated with ASA, 100 mg/d. Interestingly, in this experimental condition, NSC23766 treatment was able to further reduce the platelet aggregation in diabetic patients already treated with ASA (Figure 6C, right panel).

Discussion

We found that pharmacological inhibition of Rac1 by NSC23766 attenuated endothelial dysfunction in experimental model of diabetes mellitus and reduced platelet hyperaggregation in diabetic patients. These novel results suggest a potential protective role of Rac1 inhibition on vascular injury and platelet hyperaggregation in diabetes mellitus.

Rac1 is a regulatory component of Nox, which represents 1 of the major sources of ROS in the vascular wall. ROS generation is crucially involved in diabetes mellitus and diabetic complications.28 Although many sources of ROS contribute to increased oxidative stress in diabetes mellitus (direct effect of hyperglycemia, mitochondria, and xanthine oxidase), several Nox isoforms have been found specifically upregulated in vascular wall in the presence of high glucose.29, 30 We have previously shown that Rac1 represents a crucial modulator of ROS‐induced vascular dysfunction in preclinical model of diabetes mellitus.19 Its inhibition by an adenoviral vector carrying Rac1 dominant negative mutant protects from endothelial dysfunction in experimental diabetes mellitus.19 In the past decade, NSC23766 was identified as a small‐molecule inhibitor of Rac–guanine nucleotide exchange factor–mediated activation of Rac1.14, 15 Until now, its main field of application has been cancer biology, in which Rac1 has been reported as a novel important therapeutic target in several type of malignancies.31, 32, 33, 34, 35 Given the growing importance of Rac1 in the cardiovascular system, recently NSC23766 has been tested in different cardiovascular disorders.22

Herein, we have shown the protective effects of Rac1 inhibitor, NSC23766, on endothelial function after high glucose–induced vascular damage. Mechanistically, we demonstrated that amelioration of endothelial function via restoration of eNOS phosphorylation by Rac1 inhibition requires ROCK1 as a crucial component of Rac1 signaling in both isolated cells and vessels. Although previous studies have suggested a reduction in eNOS expression after Rac1 inhibition,36 the modulation of Rac1 activity by NSC23766 in our study was not related to changes in its expression in both mouse vessels and human endothelial cells. Moreover, we were recently able to demonstrate that Rac1 inhibition by NSC23766 exerts a beneficial effect also in human vessels, ameliorating endothelial dysfunction.16 These results pointed out the important role of Rac1 as a therapeutic target to improve vascular homeostasis.

Levay et al27 have demonstrated an effect of NSC23766 as nonselective competitive antagonist of muscarinic acetylcholine receptors in neonatal rat cardiomyocytes. In our experimental model on resistance vessels, we were able to show that NSC23766 per se did not interfere with acetylcholine vasorelaxation (Figure S5). Consistently with other studies, we have demonstrated that the activation of Rac1 (Rac1‐GTP) negatively modulates eNOS phosphorylation through ROCK‐1 pathway.37, 38 In addition to its effect on eNOS phosphorylation, inhibition of Rac1 also blunted Nox ROS production. Hence, these data demonstrated that the amelioration of endothelial relaxation obtained by Rac1 inhibition in presence of high glucose levels depends on 2 mechanisms: the increased eNOS phosphorylation and the reduction of oxidative stress.

These data prompted us to explore the effects of Rac1 inhibitor in a mouse model of diabetes mellitus. NSC23766 treatment reduced the enhanced Rac1 activation observed in preclinical diabetes mellitus and restored acetylcholine‐evoked vasorelaxation. Interestingly, amelioration of endothelial function observed after Rac1 inhibition in vessels from diabetic mice was present up to 96 hours after NSC23766 systemic administration and, accordingly, we observed a sustained inhibition of Rac1 activity in mice vessels at the same time point (Figure S6). Reduction of ROS production in diabetic vessels treated with NSC23766 can be attributed to reduction in Nox activity. Although, given the complexity of ROS production in diabetes mellitus, previously discussed, it is likely that Nox might not be the only target of Rac1 inhibition. Using pharmacological inhibition of the different Nox isoform, we were able to identify Nox4 as a critical component of NSC23766‐mediated ROS suppression in vascular wall. These data will serve as a platform to pursue more in‐depth mechanistic insights in Rac1/Nox4 interaction.

In addition to vascular damage, diabetes mellitus is also characterized by platelet dysfunction. Previous studies reported that high levels of glucose were able to increase platelet aggregation in vitro and were associated with ROS production.39 Rac1 is involved in platelet actin cytoskeleton reorganization during platelet activation. A crucial feature of platelets is represented by their ability to produce NO. Our data demonstrate that high glucose levels activate Rac1 in platelets, and this effect is associated with an impaired NO release. Notably, similar to what was observed in vessels and endothelial cells, the administration of NSC23766 in platelets protects from the deleterious effect of high glucose on NO metabolism, enhancing eNOS phosphorylation, through ROCK‐1 inhibition, and blunting oxidative stress. It is well known that a reduction in NO release is coupled with alteration in platelet activation. In this regard, 1 of the most striking changes that occur during platelet activation is the translocation of CD62 (P‐selectin) protein to the outer platelet membrane. Therefore, the expression of CD62 on the membrane of platelets is considered to be a valuable indicator for platelet activation in different diseases, including diabetes mellitus.40, 41 To evaluate the effects of NSC23766 on platelet activation in diabetes mellitus, we performed the immunoblot analysis of CD62 expression in cytosol and membrane subcellular fraction of platelets isolated from diabetic (streptozotocin‐treated) and control mice in presence and absence of NSC23766. As shown in Figure S7, membrane expression of CD62 in diabetic platelets was significantly increased. More important, in vivo administration of NSC23766 in diabetic mice abolished CD62 membrane translocation in platelets, demonstrating a significant reduction of platelet activation on Rac1 inhibition. These data suggest that the effects of NSC23766 on platelet function in diabetes mellitus are potentially beyond the expected cytoskeleton reorganization and might contribute to the beneficial effects observed in vitro and in vivo, corroborating our previous results.

Interestingly, herein we have shown that Rac1 is responsive to high glucose levels, enhancing platelet aggregation, acknowledging Rac1 as an important regulator of platelet function during hyperglycemic conditions. We demonstrated that a condition mimicking hyperglycemia in vitro increases platelet Rac1 activation. On the basis of this result, we hypothesized that increased active Rac1 platelet levels under hyperglycemic conditions could contribute to the increased platelet activity observed in diabetes mellitus. Using a specific inhibitor of Rac1 activity, NSC23766, we demonstrated that glucose‐induced platelet hyperaggregation was reduced. The increased efficacy of NSC23766 to inhibit platelet aggregation in hyperglycemic state could be explained by increased Rac1 GTP levels under this condition. The effect of glucose on platelet aggregation starts to appear for concentration of glucose of 25 mmol/L and seems to be sustained even with higher concentration (50 mmol/L), suggesting the presence of a threshold for glucose‐induced hyperaggregation in human platelets. Under normal glucose condition, the dose of 30 μmol/L, NSC23766 was sufficient to reduce Rac1 activity, modulating, in turn, platelet function. Although the dose used in our study was significantly lower compared with the one used elsewhere,42 we cannot completely exclude that the potential off‐target effects of NSC23766 might affect our results. Because the effect of NSC23766 on platelet aggregation, under high glucose condition, is already present for lower NSC23766 doses (15 μmol/L), this raised the question of how to separate the effect of hyperglycemia and the effect of NSC23766 on platelet aggregation. In our experiments, we chose relatively high levels of glucose (25 and 50 mmol/L) to mimic hyperglycemic conditions. Indeed, these levels of hyperglycemia are clinically relevant, because blood glucose commonly increases at ≈30 mmol/L in diabetic ketoacidosis and can reach 60 mmol/L during diabetic hyperosmolar coma.43 Therefore, during hyperglycemic condition, even a small increase in platelet aggregation can significantly worsen the clinical conditions. In our experiments, we recorded an increase of ≈10% in platelet aggregation on hyperglycemic stimuli using light transmission aggregometry. This increase is consistent with previous literature using the same technique,39 although it can be smoothened by the fact that the baseline percentage of platelet aggregation in our subjects is higher compared with previous reports. We speculated that this effect can be attributable to the fact that the control population is not represented by completely “healthy” subjects because, despite being free from cardiovascular disease and diabetes mellitus, they present some cardiovascular risk factors, such as hypertension and smoking, that can contribute to the increased platelet reactivity. Collectively, data from other groups and we suggest that hyperglycemic condition produces a 10% to 20% increase in platelet aggregation after stimulation with proaggregating stimuli (ie, collagen) that can reflect, at least in part, the prothrombotic state observed in diabetic patients.

Given the known off‐target effect of NSC23766,42 it is possible to speculate that glucose can prime platelets, making them more susceptible to the effects of NSC23766. Accordingly, in our model, Rac1 GTP is specifically induced by high glucose in platelets, without osmotic effects, as demonstrated by the absence of Rac1 activation with mannitol; therefore, the mild effects of NSC23766 on platelet aggregation observed in normoglycemic state might be caused by the low levels of Rac1 GTP in platelets. Accordingly, platelets from diabetic patients showed a positive correlation between activated Rac1 and levels of glycated hemoglobin. To provide further support that Rac1 is a critical target in diabetic platelets, we used another Rac1 inhibitor, called EHT1864. Differently from NSC23766, which prevents the conversion of Rac1‐GDP to Rac1‐GTP by competitively blocking the binding loop of Rac1‐specific guanine nucleotide exchange factors, EHT1864 is a specific allosteric inhibitor of the Rac family, resulting in dissociation of nucleotides.44 Using 2 different Rac1 inhibitors, we further corroborate the notion that Rac1 plays a major role in platelet proaggregating status in diabetes mellitus, underlying the peculiar platelet characteristics of diabetic subjects.

It is important to underline that in condition of Rac1 hyperactivation (such as platelets from diabetic patients), a higher dose of Rac1 inhibitor was necessary to reduce platelet aggregation compared with platelets from subjects without diabetes mellitus, pointing out that diabetes mellitus affects platelet function in many ways, which are to be exclusively recapitulated by increasing the concentrations of glucose in vitro. We also found NSC23766 treatment further reduced platelet aggregation in those subjects taking ASA, identifying Rac1 inhibition as a potential future pharmacological strategy to limit platelet hyperaggregation in diabetes mellitus. Unlike diabetic patients without ASA in whom we were able to recognize a dose‐response curve of Rac1 inhibitory effects on platelet aggregation, the additive effects of NSC23766 on ASA‐treated diabetic platelets were not dose dependent. These effects could be attributed to the lower responsiveness of platelets already treated with ASA. In fact, inhibition of platelet aggregation by other mechanisms (such as cyclooxygenase‐dependent mechanisms) could blunt the incremental inhibitory effect of Rac1 inhibition.

Study Limitations

The beneficial effects of Rac1 inhibition on hyperglycemia‐induced vascular and platelet dysfunction observed ex vivo need to be confirmed in ad hoc preclinical models of thrombus formation and using tissue‐specific genetic approaches to distinguish between platelet‐ and endothelial cell–driven effects. Nevertheless, the current findings suggest that Rac1 inhibition may be accomplished directly in vivo because of the favorable safety profile of NSC23766, as observed in our mouse model of streptozotocin‐induced diabetes mellitus. Finally, the effects of LY27632 as ROCK1 inhibitor can be also attributed, in part, to the inhibition of ROCK2, although recent studies have demonstrated that ROCK1 and ROCK2 have distinct nonredundant functions and have different targets in different cell types.45, 46

Conclusions

The results of our study address an important challenge in biological features of diabetes mellitus (namely, platelet hyperreactivity and endothelial dysfunction). We provide further evidence about the involvement of Rac1 in both vascular injury and platelet hyperaggregation induced by diabetes mellitus. Our findings could support the use of Rac1 inhibition by NSC23766 in combination with ASA for antiplatelet therapy in diabetes mellitus. Although proposing Rac1 inhibitor as an immediate therapeutic approach in diabetic patients is far beyond the scope of our work, we believe that the identification of targets/drugs able to modulate >1 function in this disease could represent the right way to move forward. In particular, the combination of beneficial effects of Rac1 inhibition on both vascular and platelet function in diabetes mellitus might represent a potential effective strategy (in the future) to increase the therapeutic compliance of these patients.

Sources of Funding

This study was supported in part by Programma Operativo Nazionale–Ricerca e Competitività 2007–2013 “Cardiotech‐tecnologie avanzate per l'innovazione e I'ottimizzazione dei processi, diagnostici terapeutici e di training dedicati alla gestione clinica, interventistica e riabilitativa dei paziente affetti da sindromi coronariche acute” (PON01_02833; Esposito).

Disclosures

None.

Data S1. Supplemental methods.

Table S1. Characteristics of Patients

Table S2. Characteristics of diabetic mice used in the study.

Figure S1. Nitroglycerine‐evoked vasorelaxation in pre‐constricted mesenteric arteries (n=4 for each group) treated with low (Glu 5 mmol/L, full circles) and high glucose levels (Glu 25 mmol/L, empty circles).

Figure S2. A, Representative immunoblot (left) and densitometric analysis (right) of p‐eNOS, eNOS, ROCK1, Rac1‐GTP, Rac1 and β‐actin in serum‐starved HUVECs treated with 5 or 25 mmol/L of glucose, stimulated with acethylcholine. Cells have been treated with NSC23766 alone, with LY27632 alone or concomitantly with both compound. The data are presented as mean±SEM from three independent experiments.*P<0.05 vs all. B, Effects of glucose and Rac1 inhibitor treatment on NADPH oxidase activity in HUVECs. Levels of NADPH activity was measured by lucigeninenhanced chemiluminescence. Four independent experiments. *P<0.05 vs all.

Figure S3. A, Quantification of platelets aggregation presented as percentage of light transmission of platelets from control subjects treated with increasing concentrations of mannitol. Data are expressed as mean±SEM. n=4 independent experiments from individual subjects. B, Quantification of platelets aggregation presented as percentage of light transmission of platelets from control subjects treated with manitol plus increasing concentrations of NSC23766. Data are presented as mean±SEM. n=4 independent experiments from individual subjects. C, Representative immunoblot of Rac1 and Rac1‐GTP levels in platelets from control subjects (CTRL) treated with mannitol 5 and 25 mmol/L plus NSC23766, (D) and relative densitometric analysis. n=4 independent experiments from individual subjects.

Figure S4. Quantification of platelets aggregation presented as percentage of light transmission of platelets from control (CTRL) and diabetic (Db) subjects treated with two different dose of EHT1864. n=4 independent experiments from individual subjects. *P<0.05 vs CTRL 0 (without EHT1864), # P<0.05 vs Db 0 (without NSC23766).

Figure S5. Acetylcholine (ACh) vasorelaxation in preconstricted mice mesenteric arteries in basal condition (Ctrl, full circle) and after i.p. treatment with Rac1 inhibitor, NSC23766 5 mg/kg, at different timepoints (6–12–24–48 hours) (n=4 for each group).

Figure S6. Representative immunoblot (left) anddensitometric analysis (right) of Rac1‐GTP, total Rac1 and β‐Actin levels in mesenteric arteries from streptozotocin‐treated mice injected i.p. with NSC23766 (5 mg/kg) and evaluated at different timepoints (12, 48, 96 hours). n=3 independent experiments. *P<0.05 vs STZ+NSC23766 (12 hours); # P<0.05 vs STZ+NSC23766 (48 hours); § P<0.05 vs STZ+NSC23766 (96 hours).

Figure S7. Representative immunoblots (left) and densitometric analysis (right) showing protein levels of CD62, Na/K ATPase, and β‐Actin in subcellular fractions (cytosol and membranes) of platelets isolated from STZ‐ and vehicle‐ treated mice in presence or absence of NSC23766. Na/K ATPase and β‐Actin were used as membrane and cytoplasmic markers, respectively.

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