10.4081/btvb.2022.19
Aim of the present study was to compare the lipid-lowering, antithrombotic and antiatherogenic properties of NCX-6550, nitropravastatin, a nitric-oxide donating derivative of pravastatin, with those of pravastatin in hypercholesterolemic mice. LDL receptor-deficient mice (LDLR–/–) on a normal diet (ND) showed enhanced cholesterol levels as compared to wild type (WT) mice (6.8±1.2 mmol/L and 2.8±0.82 mmol/L, respectively). High fat diet (HFD) induced a large enhancement of cholesterolemia in LDLR–/– mice (23.7±5.7 mmol/L, p&lt;0.0001 vs LDLR–/– ND and WT mice. Treatment with NCX 6550 (48 mg/kg), but not with equimolar pravastatin, reduced cholesterol in LDLR–/–HFD. Platelet adhesion to collagen under high shear rate (3000 sec–1) was significantly higher in LDLR–/– than in normal mice, and further enhanced in LDLR–/–HFD (-27%, p&lt;0.0001 vs untreated). NCX 6550 (48 mg/kg), but not pravastatin, reduced platelet adhesion, especially in LDLR–/–HFD. U46619-induced platelet aggregation ex vivo was also inhibited by NCX 6550 (48 mg/kg) but not by the parent compound. Finally, photochemically-induced acute (1 hr) femoral artery thrombosis and delayed (21 days) intimal thickening was assessed. Thrombus size was larger in LDLR–/– on HFD than in normocholesterolemic mice (0.46±0.04 vs 0.18±0.08 mg) and it was reduced by NCX 6550 (48 mg/kg) (0.08±0.02 mg, p&lt;0.0001), but not by pravastatin (0.4±0.01 mg p=NS). Intimal thickening was greater in hypercholesterolemic than in normal mice (I/M normal=0.53±0.16, LDLR–/–=1.1±0.15, LDLR–/–HFD=1.75 ±0.25). Both NCX 6550 and pravastatin reduced intimal thickening in normal (-95% and -74.5%, respectively) and LDLR–/– mice (-98% and -91%), while in strongly hyperlipidemic animals (LDLR–/–HFD) NCX 6550 was more effective than pravastatin (-98% vs -65%, p&lt;0.0001). NCX 6550 shows greater antithrombotic and antiatherogenic activity than pravastatin in highly hypercholesterolemic mice.
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Bleeding, Thrombosis and Vascular Biology
Nitric oxide
A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice
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Bleed Thromb Vascul Biol
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Gresele P, Marzotti S, Guglielmini G, et al. Hyperglycemia-induced platelet activation in type 2 diabetes is resistant to aspirin but not to a nitric oxide-donating agent. Diabetes Care 2010;33:1262-8.
A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice
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                                                                                                          ORIGINAL ARTICLE

A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic
activity in mice
Stefania Momi,1 Giuseppe Guglielmini,1 Giulia Ciarrocca Taranta,1 Elisa Giglio,1 Angela Monopoli,2 Paolo Gresele1
1
 Division of Internal and Cardiovascular Medicine, Department of Medicine and Surgery, University of Perugia, Italy; 2NicOx Research
Institute, Milan, Italy



ABSTRACT
     Aim of the present study was to compare the lipid-lowering, antithrombotic and antiatherogenic properties of NCX-6550,
nitropravastatin, a nitric-oxide donating derivative of pravastatin, with those of pravastatin in hypercholesterolemic mice. LDL
receptor-deficient mice (LDLR–/–) on a normal diet (ND) showed enhanced cholesterol levels as compared to wild type (WT) mice
(6.8±1.2 mmol/L and 2.8±0.82 mmol/L, respectively). High fat diet (HFD) induced a large enhancement of cholesterolemia in LDLR–/–
mice (23.7±5.7 mmol/L, p<0.0001 vs LDLR–/– ND and WT mice. Treatment with NCX 6550 (48 mg/kg), but not with equimolar
pravastatin, reduced cholesterol in LDLR–/–HFD. Platelet adhesion to collagen under high shear rate (3000 sec–1) was significantly
higher in LDLR–/– than in normal mice, and further enhanced in LDLR–/–HFD (-27%, p<0.0001 vs untreated). NCX 6550 (48 mg/kg),
                                                                        but not pravastatin, reduced platelet adhesion, especially in




                                                                                               ly
                                                                        LDLR–/–HFD. U46619-induced platelet aggregation ex vivo
                                                                        was also inhibited by NCX 6550 (48 mg/kg) but not by the




                                                                                        on
   Correspondence: Stefania Momi, Division of Internal and Cardio-      parent compound. Finally, photochemically-induced acute (1
   vascular Medicine, Department of Medicine and Surgery, Univer-       hr) femoral artery thrombosis and delayed (21 days) intimal
   sity of Perugia, Strada Vicinale Via delle Corse – Sant’Andrea delle thickening was assessed. Thrombus size was larger in LDLR–/–



                                                                                   e
   Fratte, 06132 Perugia, Italy.
                                                                        on HFD than in normocholesterolemic mice (0.46±0.04 vs
   Tel.: +39.075.5783989.                                                     us
   E-mail: stefaniamomi@gmail.com
                                                                        0.18±0.08 mg) and it was reduced by NCX 6550 (48 mg/kg)
                                                                        (0.08±0.02 mg, p<0.0001), but not by pravastatin (0.4±0.01
   Key words: NCX 6550; Intimal hyperplasia; Thrombosis; Platelets;     mg p=NS). Intimal thickening was greater in
                                                                              al
   Nitric oxide.                                                        hypercholesterolemic than in normal mice (I/M
                                                                        normal=0.53±0.16, LDLR–/–=1.1±0.15, LDLR–/–HFD=1.75
                                                           ci

   Acknowledgments: The Authors thank NiCox SA for kindly pro-          ±0.25). Both NCX 6550 and pravastatin reduced intimal
   viding pravastatin and NCX 6550 and for partial support to some
                                                         er


                                                                        thickening in normal (-95% and -74.5%, respectively) and
   of these studies.
                                                                        LDLR–/– mice (-98% and -91%), while in strongly
                                                        m




   Contributions: SM and PG designed the study and wrote the man-
                                                                        hyperlipidemic animals (LDLR–/–HFD) NCX 6550 was more
   uscript; SM performed in vivo experiments, analyzed data, and ed-    effective than pravastatin (-98% vs -65%, p<0.0001). NCX
                                            om




   ited the manuscript; GCT, GG and EG performed experiments and        6550 shows greater antithrombotic and antiatherogenic activity
   analyzed data; GG analyzed data; AM revised the manuscript.          than pravastatin in highly hypercholesterolemic mice.
                                      -c




    Conflict of interest: NCX 6550 was produced by NicOx Research
    Institute. NicOx Research Institute partially support some of these
                             on




    studies. Angela Monopoli was previously an employee at NicOx
    Research Institute, Milan, Italy.                                          INTRODUCTION
                                                                                   Atherosclerosis is a chronic inflammatory process and
                       N




    Funding: This work was supported in part by a grant from
    CARIPLO (#2018.0483) to PG.                                                inflammation plays an important role in the development
                                                                               of cardiovascular (CV) disease.1 In mice, atherogenesis
    Received for publication: 9 February 2022.
                                                                               may be triggered by raising plasma cholesterol levels and
    Accepted for publication: 6 May 2022.
                                                                               to this end LDL receptor-deficient mice (LDLR–/–) fed a
    Publisher’s note: All claims expressed in this article are solely those    high fat/high cholesterol diet represent a commonly used
    of the authors and do not necessarily represent those of their affili-     model of atherosclerosis.2 Indeed, young LDLR–/– mice
    ated organizations, or those of the publisher, the editors and the re-
                                                                               on a normal diet are characterized by an only slightly el-
    viewers. Any product that may be evaluated in this article or claim
    that may be made by its manufacturer is not guaranteed or endorsed         evated plasma cholesterol, a phenomenon attributable to
    by the publisher.                                                          the presence of an alternative ApoB-II pathway for LDL
                                                                               clearance in the mouse. Male LDLR–/– mice fed a west-
     Copyright: the Author(s), 2022
                                                                               ern-type diet additioned with 1% cholesterol, without
    ©

    Licensee PAGEPress, Italy
    Bleeding, Thrombosis and Vascular Biology 2022; 1:19                       cholate, instead display a strong elevation of LDL cho-
    doi:10.4081/btvb.2022.19                                                   lesterol and total cholesterol that reaches ~25 mmol/L and
                                                                               extensive atherosclerosis3 and represent thus a good
    This work is licensed under a Creative Commons Attribution-
    NonCommercial 4.0 International License (CC BY-NC 4.0).
                                                                               model to study lipid lowering agents and drugs potentially
                                                                               preventing atherosclerosis progression.

                                                Bleeding, Thrombosis and Vascular Biology 2022; 1:19
6                                                          S. Momi et al.




    Hyperlipidemia in humans is characterized by en-                 of hybrid drugs combining a statin with a NO-donating
hanced oxidative stress, generation of biologically active           moiety has been developed and shown to exert additional
oxidized lipids and platelet hyperreactivity which con-              properties in vitro and in vivo as compared to the parent
tribute to the enhanced CV risk.4 LDLR–/– mice on a high-            compounds, such as enhanced anti-inflammatory and an-
fat diet have been shown to recapitulate human                       tiproliferative activities.18,19 antiplatelet and antithrom-
hyperlipidemia findings with the accumulation of oxi-                botic activities20 and a pro-healing effect related to
dized lipids and in particular of the atherogenic oxidized           potentiation of vascular regeneration in a mouse model of
choline glycerophospholipids (oxPCCD36), in serum, in-               limb ischemia.21 Moreover, several previous observations
creased platelet reactivity through the interaction between          show that NO-donating agents, such as the NO-donating
oxPCCD36 and platelet CD36 and the development of a pro-             aspirin NCX 4016 reduce the onset of diabetes by com-
thrombotic phenotype.5                                               bining antioxidant, anti-platelet activation and endothelial
    Inflammation is important in the pathogenesis of ather-          protective properties.22–24
osclerotic disease since atherosclerotic plaques are strongly            We have previously shown that the addition of a NO-
infiltrated by activated immune cells and atherosclerosis            donating moiety to atorvastatin, a lipophilic statin, enhances
associates with enhanced circulating levels of inflammatory          its effectiveness in models of in vivo peroxidation and
biomarkers, such as interleukin-6 (IL-6) or tumor necrosis           strong atherosclerosis-related inflammation.16 Considering
α (TNF-α).6 IL-6 is a multifunctional cytokine with en-              that the hydrophilic pravastatin was reported to be less di-




                                                                                              ly
docrine and paracrine effects, which mediates several func-          abetogenic compared with other statins25 and that its anti-




                                                                                      on
tions in host defense, but also promotes atherogenesis,              inflammatory and anti-atherogenic effect is magnified
dyslipidemia, hypertension and insulin resistance through            when combined with an antiplatelet drug,15 we focused our
activated macrophages and lymphocytes. It is also an active          work on a new hybrid drug combining NO with pravastatin



                                                                                 e
signaling molecule synthesized in adipose tissue.7                   (NCX 6550) with the aim of characterizing its antiplatelet,
    Statins are widely used in the treatment of cardiovas-
                                                                            us
                                                                     antithrombotic and anti-atherosclerotic effects.
cular disease associated with atherogenic dyslipidemia. In
addition to their potent action in cholesterol-rich lipopro-
                                                                    al

teins, statins have pleiotropic properties that may contribute       MATeRIAls AND MeThODs
                                                        ci

to their beneficial effects on cardiovascular diseases.8 Many        Mice
of the beneficial pleiotropic effects of statins occur as a re-
                                                      er



sult of improved endothelial function and reduced inflam-                Wild-type C57BL/6J male mice were purchased from
                                                                     Charles River Laboratories (Lecco, Italy). LDLR–/– mice
                                                     m




matory processes.9 Despite their therapeutic efficacy,
monotherapy with statins alone is often insufficient to              were purchased from The Jackson Laboratories.26 LDLR–/–
                                           om




achieve therapeutic goals in patients with atherosclerosis.          mice had been backcrossed 10 generations onto the
    Hypercholesterolemia-induced vascular disease is                 C57BK6/J background. Groups of male mice were placed
characterized by the loss of the physiological properties            on the different diets after weaning at 4 weeks of age.
                                      -c




of endothelium, known as endothelial dysfunction.10,11 En-               LDLR–/– mice were genotyped by using three primer
dothelial dysfunction is an early event in the pathogenesis          sets that specifically amplified the wild-type or the knock-
                              on




of atherosclerosis and is essentially due to diminished pro-         out LDLR allele (forward: 5’- CCA TAT GCA TCC CCA
duction/availability of nitric oxide (NO), decreased eNOS            GTC TT-3’; reverse: 5’- GCG ATG GAT ACA CTC ACT
                         N




expression, eNOS uncoupling and enhanced NO degra-                   G C-3’; primer sequence used for NEO detection: 5’- AAT
dation.12 Endothelial- and platelet-derived NO inhibits in-          CCA TCT TGT TCA ATG GCC GAT C-3’).
flammatory cell activation thus exerting a potential                     The study was approved by the Committee on Ethics
antiatherogenic activity.13 Statins, despite their ability to        of Animal Experiments of the University of Perugia and
enhance NO-production and bioavailability by increasing              by the Italian Ministry of Public Health (Authorization n°
eNOS mRNA stability blocking the geranylgeranylation                 49/2004-B).
of the GTPase Rho,14 may not be able to normalize en-
                                                                     Diet
dothelial function in severe hypercholesterolemia or in
acute coronary syndromes. The activity of statins may be                 C57BL6/J male mice were maintained under normal
potentiated by their combination with molecules of                   diet (ND) (Mucedola, Milan, Italy). LDLR–/– male mice
proven anti-inflammatory activity, such as cilostazol, a             were maintained under normal chow until 4 weeks of age
phosphodiesterase inhibitor, or nitric oxide donors.15,16            and then they were randomly divided in two groups: one
    Moreover, in the last few years, a growing body of ev-           maintained under normal chow, and one switched to a
idence has highlighted a 10-12% increase in new-onset                high fat diet (HFD) (Tekland Custom Diet, Wisconsin,
diabetes mellitus in hypercholesterolemic patients on                diet #TD 95286: 21% [wt/wt] fat, 1% cholesterol, 19.5%
statin therapy.17 Therefore, in the last years, a new class          casein, and no sodium cholate) for 20 weeks. Drug treat-

                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:19
                A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice              7



ment was initiated starting from the 16th week of the dif-            After one hour at 37°C serum was retrieved by centrifu-
ferent diet regimens.                                                 gation at 1000×g for 15 min at room temperature. Total,
                                                                      LDL, HDL cholesterol and triglycerides were measured
Drugs                                                                 by a commercial colorimetric method (Menarini Pharma-
                                                                      ceuticals, Firenze, Italy).16 LDL cholesterol was calculated
    Mice were administered pravastatin (40 mg/kg), a
                                                                      from total and HDL cholesterol and triglyceride levels.
pravastatin hybrid donor-NO compound (NCX 6550) (48
mg/kg) or their vehicle by oral route (gavage) once a day
                                                                      Photochemical injury-induced femoral artery
for 3 or 4 weeks, depending on the end point of the ex-
                                                                      thrombosis
periment.
    NCX 6550 and pravastatin were dissolved in DMSO                        Mice were anesthetized with sodium pentobarbital (80
(2%) and then diluted in PEG 400 for oral gavage. The                 mg/kg, i.p.) and a butterfly 25G needle was inserted in one
concentration of DMSO in the administered mixture                     of the tail veins for rose Bengal infusion. The right femoral
never exceeded 1%. The doses of the drugs used were                   artery was surgically exposed and transilluminated with
equivalent on the basis of the respective molecular                   green light (wavelength 540 nm), by a xenon lamp with a
weights (NCX 6550/pravastatin: 1.2/1) and contained the               heat-absorbing filter, through an optic fiber positioned 5
same amount of pravastatin.                                           mm away from the arterial segment (Hamamatsu Photon-




                                                                                        ly
                                                                      ics, Shizuoka, Japan). Green light irradiation was protracted
Platelet aggregation                                                  for 25 min; the infusion of rose Bengal (20 mg/kg) started




                                                                                on
                                                                      5 min after the beginning of irradiation and lasted for 5 min.
    Blood was collected by cardiac puncture in 4%                     Blood flow was recorded by a perivascular flow probe (0.5
trisodium citrate (1:10, v/v) from anesthetized LDLR–/–               Transonic System) connected to a flowmeter (Transonic


                                                                           e
mice (blood was pooled from at least 5 mice for each                  System Inc, model T402, Ithaca, NY, USA) and interpreted
treatment group) 1h after the last administration of NCX
                                                                    usby a computerized data acquisition program (Biopac Sys-
6550 (48 mg/kg, o.i.d.), pravastatin (40 mg/kg, o.i.d.) or            tem Inc, Santa Barbara, CA, USA). Time to occlusion of
vehicle. Platelet-rich plasma (PRP) was obtained by cen-
                                                            al
                                                                      the femoral artery was measured. One hour after the end of
trifugation of blood at 150×g for 15 min at room temper-              irradiation, the femoral artery was excised and the thrombus
                                               ci

ature. Light transmission aggregometry was carried out                was weighed after rinsing, blotting on filter paper and dry-
using a four-channel aggregometer (Aggregocorder,
                                             er


                                                                      ing overnight at 60°C.16,29–31
Menarini, Florence, Italy), using the thromboxane (TxA2)
                                            m




analogue U46619 (3 µM) (MM3), collagen (2 µg/ml) and                  Femoral arteries atherogenesis
ADP (3 µM) as aggregating agents (n=3 experiments, 8
                                  om




animal per group) as previously described.27                              To evaluate femoral artery intima hyperplasia triggered
                                                                      by photochemical injury, the wound was closed and ani-
Platelet adhesion to collagen under flow                              mals were returned to their cages. Drugs were administered
                             -c




                                                                      once a day for 21 days by gavage starting one hour before
     Platelet adhesion to a collagen-coated surface under             the induction of femoral artery damage. One hour after the
                     on




flow conditions was studied according to published proto-             last drug administration mice were anesthetized, the chest
cols.16,28 Briefly, citrated blood samples (0.5 ml) were              and abdominal cavities were opened and a catheter was in-
                N




passed through a rectangular parallel plate perfusion cham-           serted into the left ventricle and circulation was first washed
ber over a plastic coverslip sprayed with collagen from               with saline and then with a solution of 2% glutaraldehyde
equine tendon (~ 30 µg/cm2), at a wall shear rate of 3,000            and 1% paraformaldehyde (PFA) in 0.1 mM PBS, pH 7.4,
s–1. The chamber was then perfused with 0.1 % bovine                  at physiologic pressure (90 to 100 mmHg) for 10 min. The
serum albumin in physiologic solution, pH 7.3, to remove              femoral artery was then removed and fixed overnight in 4%
all residual blood and then the coverslip was harvested,              PFA in 0.1 mM PBS. Femoral artery segments were em-
gently washed with 10 mM HEPES, pH 7.3, and fixed with                bedded in paraffin and cut consecutively in 5µm thick sec-
0.25 % glutaric-dialdehyde in PBS, pH 7.4. Adhering                   tions. Sections taken 500 µm apart, were stained with
platelets were stained with May-Grünwald/Giemsa and ob-               hematoxylin and eosin for morphometric analysis. Media
served under an optical microscope. The area covered by               and intima area were measured using a specific software
platelets was measured with a computerized image analyzer             (NIH Scion Image, Bethesda, MD, USA). Sections were
(NIH Scion Image, Frederick, Maryland).16,28                          assessed blindly as regards to treatment.16,31

serum lipids                                                          Quantification of atherosclerosis in the aorta
   Blood from mice fasted overnight was collected by                     Atherosclerotic lesions were quantified by en face
cardiac puncture into glass tubes without anticoagulant.              analysis of the whole aorta and expressed as percent of

                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:19
8                                                         S. Momi et al.




the entire surface area of the aorta covered by plaques.
For en face preparations, a cannula was inserted into the           ResUlTs
left ventricle and the aortic tree was fixed by perfusion           NCX 6550 and pravastatin reduce serum cholesterol
with ice-cold PBS containing 4% PFA, 5% sucrose and 2
μM EDTA for 10 min.16 The aorta was then opened lon-                    Serum cholesterol was enhanced in wild-type mice
gitudinally, from the heart to the iliac arteries, while still      under HFD as compared to wild-type mice on a normal diet
attached to the heart and major branching arteries. The             (5.95±0.25 vs 2.87±0.82 mmol/L, respectively, p<0.05).
primary incision followed the ventral side of the aorta and         LDLR–/– mice kept under HFD had strikingly higher serum
                                                                    cholesterol as compared with LDLR–/– mice on a normal
the inner curvature of the arch. To obtain a flat preparation
                                                                    diet (23.74±5.7 and 6.77±1.23 mmol/L, respectively,
for imaging, a second incision was made along the outer
                                                                    p<0.05) (Figure 1a). The lipid lowering effect of NCX 6550
curvature of the arch. The aorta (from the heart to the iliac
                                                                    in conditions of stronger hypercholesterolemia was not en-
bifurcation) was then removed and “pinned out” on a
                                                                    hanced as compared to equimolar pravastatin. (Figure 1b).
black wax surface in a dissecting pan using stainless steel
pins 0.15 mm in diameter. Aortas were briefly rinsed in
                                                                    hypercholesterolemia induces platelet hyperreactivity
70% ethanol; immersed for 6 min in a filtered solution
containing 0.5% Sudan IV, 35% ethanol, and 50% ace-                 Platelet aggregation
tone, and destained for 5 min in 80% ethanol. The Sudan




                                                                                              ly
                                                                        In LDLR-HFD mice platelet aggregation was signif-
IV–stained aortas were photographed for quantification              icantly enhanced as compared to normocholesterolemic




                                                                                      on
of atherosclerotic lesions.16,32                                    mice using all agonists (Figure 2a). In conditions of
                                                                    strong cholesterolemia (LDLR–/–HFD), ex vivo platelet
Circulating tumor necrosis factor-α (TNFα)                          aggregation was significantly inhibited by NCX 6550


                                                                                e
and interleukin-6 (Il-6) levels                                     treatment but not by pravastatin with all the agonists
                                                                           us
   TNFα and IL-6 were assessed in serum using com-                  used (Figure 2b).
mercial ELISA kits purchased from R&D System (Min-
                                                                   al
neapolis, MN, USA) (Quantikine Mouse TNFα                           Platelet adhesion to a collagen-coated surface ex vivo
Immunoassay, cat. #MTA00 and Mouse IL-6 Quantikine
                                                       ci

                                                                        Platelet adhesion to collagen was significantly
ELISA Kit, cat. #M6000B).16                                         enhanced in strongly hypercholesterolemic mice
                                                     er



                                                                    (Figure 2c).
Plasmatic nitrites and nitrates                                         Treatment with NCX 6550, but not pravastatin, sig-
                                                    m




    Blood was collected in trisodium citrate (4%) and               nificantly reduced platelet adhesion to collagen in normo-
                                           om




plasma was obtained by centrifugation at 1,000×g for 10             cholesterolemic wild-type (7.4%) as well as in LDLR-ND
min and nitrites and nitrates (NOx) levels were measured            mice (23%); the reduction of platelet adhesion induced by
by a colorimetric, non-enzymatic method (Oxford Bio-
                                     -c




medical Research, Rochester Hills, MI, USA), as reported
elsewhere.16,33
                              on




Plasma cGMP
                         N




    Blood was collected in trisodium citrate (4%) and
plasma was obtained by centrifugation at 1,000×g for 10
min and plasma cGMP levels were measured by ELISA
(GE Healthcare, Milan, Italy), as reported elsewhere.16,33

statistical analysis
    Data were analyzed by one way ANOVA, followed
by the Newman-Keuls multiple comparison test between
all groups. The correlation between different parameters
was assessed by the Spearman’s test. All analyses were              Figure 1. a) Sixteen weeks of high fat diet (HFD) strikingly en-
performed by the GraphPad Prism 4.00 for Windows                    hance total serum cholesterol in LDLR–/– mice. b) NCX 6550
software (GraphPad Software, San Diego, CA, USA).                   48 mg/kg significantly reduced total cholesterol levels, more
Data are expressed as arithmetic means ± SEM. A p                   than equimolar pravastatin (n=10-12 mice per group). Statistical
value of less than 0.05 was considered as statistically             analysis: ANOVA followed by the Newman-Keuls multiple
significant.                                                        comparisons test.


                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:19
                 A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice                9



NCX 6550 was even more marked in strongly hypercho-                     femoral artery: time to occlusion in wild-type, LDLR–/–
lesterolemic LDLR–/–HFD mice (36.4%) (Figure 2d).                       on ND and LDLR–/– on HFD was progressively shorter
                                                                        (Figure 3c). Besides a reduced thrombus weight ob-
effects on thrombosis                                                   served in LDLR–/– mice on HFD upon NCX 6550 ad-
                                                                        ministration, a significant enhancement of time to
Femoral artery thrombosis
                                                                        occlusion was achieved. Pravastatin slightly prolonged
   Thrombus weight of wild-type mice was 0.18±0.08                      time to occlusion (Figure 3d).
mg. In vehicle-treated LDLR–/– mice on ND thrombus
weight was 0.26±0.018 mg and it was further, signifi-                   effects on atherosclerosis
cantly, enhanced when these mice were fed a HFD for 16
                                                                        Photochemical injury-induced intimal thickening
weeks (0.46±0.08 mg) (Figure 3a). In LDLR–/– mice on
                                                                        of femoral arteries
HFD NCX 6550 (48 mg/kg) but not pravastatin, signifi-
cantly reduced thrombus weight (0.08±0.02 and                             Neointima hyperplasia was enhanced in LDLR–/– mice
0.38±0.08, respectively, n=5) (Figure 3b).                             on ND as compared with wild-type mice (I/M=1.077±0.15
   Different levels of cholesterolemia modulated not                   vs 0.53±0.16, respectively) and even more strongly in
only thrombus size but also the time to occlusion. To a                LDLR–/– mice on HFD (I/M=1.75±0.25, p<0.001 vs wild-
reduced thrombus size corresponded a longer time to oc-                type mice) (Figure 4a).




                                                                                          ly
clusion after photochemically-induced damage to the                       Both drugs, NCX 6550 and pravastatin, were able to re-




                                                                                  on
                                                                            e
                                                                     us
                                                              al
                                                ci
                                              er
                                             m
                                    om
                              -c
                       on
                  N




Figure 2. a) Ex vivo U46619-, collagen- and ADP-induced platelet aggregation in wild-type mice kept on standard diet (SD), LDLR–/–
mice on SD, and LDLR–/– mice on high fat diet (HFD). Five mice per group were used. Data are expressed as mean ± SEM of 3 experiments.
b) Effect of NCX 6550 (48 mg/kg, blue circles) and pravastatin (40 mg/kg, red circles) on U46619-, collagen and ADP-induced platelet
aggregation in LDLR–/– mice on HFD. Platelet aggregation was evaluated with platelet rich plasma. Black circles: platelets from vehicle-
treated mice. c) Ex-vivo platelet adhesion to a collagen-coated surface under flow was carried out in wild-type mice kept on SD, LDLR–/–
on SD, and LDLR–/– mice on HFD. d) Effect of 4-weeks treatment with NCX 6550 and pravastatin on platelet adhesion under shear in
HFD-fed in LDLR–/– mice. Statistical analysis: ANOVA followed by the Newman-Keuls multiple comparisons test (n=6-9 per group).


                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:19
10                                                        S. Momi et al.




duce intimal hyperplasia in wild type and in LDLR-ND                Nitric oxide and its effects
mice. Interestingly, in strongly hypercholesterolemic mice,
                                                                        The administration of NCX 6550 for 3 weeks, but not
while NCX 6550 maintained its antiproliferative activity,
                                                                    of pravastatin, enhanced significantly plasmatic NO2/NO3
pravastatin lost a significant part of its effect (Figure 4b).
                                                                    in strong hypercholesterolemic LDLR–/– mice (Figure 6a).
                                                                    Similarly, in LDLR–/– mice treated with a single adminis-
Aortic atherosclerosis
                                                                    tration of NCX 6550 (48 mg/kg, p.o.) a marked increase
    Extensive atherosclerotic lesions were observed                 of plasmatic cGMP was found (control: 6.094±4.4; NCX
throughout the aortas in LDLR–/– mice on HFD (40.1±1.1              6550: 20.52±6.2 pmol/ml, n=5, p=0.0214 vs vehicle).
% of surface covered by plaques). Treatment with NCX                Pravastatin did not change plasmatic cGMP (Figure 6b).
6550 (48 mg/kg for 3 weeks) reduced the lesion area by
65% (14.04±3.3 % of surface covered by plaques, n=5,
p<0.0001 vs control), significantly more than pravastatin           DIsCUssION
(40 mg/kg for 3 weeks) (24.3±2.1 % of surface covered
                                                                        An increased platelet reactivity and, therefore of their
by plaques, 39% reduction, n=4, p<0.001 vs control)
                                                                    thrombogenic potential, is a feature of a number of patho-
(Figure 4c,d).
                                                                    physiological conditions associated with cardiovascular
                                                                    risk factors, including dyslipidemia, diabetes and the




                                                                                             ly
Circulating inflammatory biomarkers
                                                                    metabolic syndrome.4 Indeed, it has been suggested that




                                                                                     on
    Consistent with the inflammatory condition associated           the CV risk associated with dyslipidemia may be due to
with atherosclerosis, circulating levels of the inflamma-           effects on both thrombogenesis and atherogenesis;34 thus,
tory markers TNF-α and IL-6 were significantly increased            the control of platelet reactivity is regarded to be critical



                                                                                e
in hypercholesterolemic conditions (Figure 5a,c). In                for the prevention of coronary artery disease in hyperlipi-
LDLR–/– mice on HFD 3 weeks treatment with NCX 6550
                                                                           us
                                                                    demic patients.35
reduced IL-6 and TNF-α levels. Pravastatin reduced them                 In this paper we show that increasing levels of choles-
also (Figure 5b,d).                                                 terolemia translate in progressively greater platelet hyper-
                                                                   al
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                                                                                   Figure 3. Effect of cholesterolemia on photo-
                                                                                   chemically-induced thrombosis in mice kept on
                                                                                   SD, LDLR–/– on SD, and LDLR–/– mice on HFD
                                                                                   on thrombus weight (a) and on time to occlusion
                                                                                   (c). Pravastatin and, even more, NCX 6550, re-
                                                                                   duced thrombus weight (b) and time to occlusion
                                                                                   (d) in LDLR–/– mice on high fat diet (HFD) (n=5
                                                                                   per treatment). Statistical analysis: ANOVA fol-
                                                                                   lowed by the Newman-Keuls multiple compar-
                                                                                   isons test (n=6-9 per group).


                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:19
A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice                11




                                                                 Figure 4. a) Thickening of intimal/media of the
                                                                 femoral artery induced by photochemical damage is
                                                                 enhanced in strongly LDLR–/– hyperlipidemic mice




                                                                        ly
                                                                 than in LDLR–/– and wild-type mice on SD. b) NCX
                                                                 6550 significantly reduced I/M thickness more than its




                                                                on
                                                                 parent compound and this effect was more marked in
                                                                 strong hypercholesterolemia. c) Hyperlipidemia-in-
                                                                 duced aortic lipid plaque deposition in total aorta was




                                                           e
                                                                 significantly reduced by pravastatin treatment and,
                                                    us           even more, by NCX 6550. d) Representative photomi-
                                                                 crographs of en face Sudan IV staining of total aorta.
                                                                 Statistical analysis: ANOVA followed by the Newman-
                                            al
                                                                 Keuls multiple comparisons test (n=6-9 per group).
                               ci
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                  om
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N




                                                                 Figure 5. IL-6 and TNFα levels in wild-type mice
                                                                 kept on a standard diet (SD), LDLR–/– mice on a SD,
                                                                 and LDLR–/– mice on a high fat diet (HFD) (a,c). Ef-
                                                                 fect of pravastatin or NCX 6550 on the levels of
                                                                 serum IL-6 (b) and TNFα (d) of LDLR–/– mice kept
                                                                 on a HFD. Statistical analysis: ANOVA followed by
                                                                 the Newman-Keuls multiple comparisons test (n=6-
                                                                 9 per group).


                      Bleeding, Thrombosis and Vascular Biology 2022; 1:19
12                                                      S. Momi et al.




reactivity in mice which associates with a marked pro-            occur in humans during the progression of atherosclerosis
thrombotic phenotype. The response of platelets from              and which may also contribute to plaque disruption.36
strongly hypercholesterolemic mice to platelet agonists           These processes are strongly exacerbated by hypercholes-
was enhanced as compared to platelets from normocho-              terolemia. In fact, in hypercholesterolemic animals the
lesterolemic or mildly hypercholesterolemic LDLR–/–               time to thrombus formation was significantly shortened
mice. Moreover, platelet adhesion to collagen under high          and thrombus size enhanced. Interestingly, NCX 6550 sig-
shear rate, a model that reproduces the conditions occur-         nificantly prevented thrombosis in these conditions, while
ring within stenotic coronary arteries,36 was also enhanced       pravastatin was ineffective.
in strong hypercholesterolemic mice. Interestingly, the               Hypercholesterolemia is associated with a low-grade
NO-donor pravastatin hybrid drug NCX 6550, but not                inflammatory state;35 this is partly due to the generation
pravastatin, was able to inhibit both platelet aggregation        of reactive oxygen species that induce lipid peroxidation
and platelet adhesion to collagen in strongly hypercholes-        of cell-membrane phospholipids and of circulating LDL
terolemic mice, an observation of particular interest con-        and these may participate in potentiating platelet activa-
sidering that aspirin, the conventional antiplatelet agent        tion,39 thus contributing to platelet hyperreactivity. More-
used for CV prevention, is not able to prevent platelet ad-       over, the enhanced release of reactive oxygen species
hesion in this model.33                                           contributes to the scavenging of nitric oxide and to the re-
    NCX 6550 and pravastatin displayed a similar lipid-           duction of its bioavailability.40 In this model NCX 6550




                                                                                            ly
lowering activity, but only NCX 6550 was able to exert            successfully prevented atherosclerosis and inflammation




                                                                                   on
an antiatherogenic and anti-inflammatory activity, likely         while pravastatin was significantly less effective. This
due to the released nitric oxide. The anti-inflammatory ac-       suggests that in conditions of severe endothelial injury as-
tivity of NCX 6550 was previously shown also in human             sociated with the generation of oxygen radicals the direct



                                                                              e
monocyte/macrophages where NCX 6550 was more ef-                  supply of NO allows to attain a significant anti-athero-
fective than pravastatin in inhibiting PMA-evoked release
of TNF-α and IL-6.37
                                                                         us
                                                                  sclerotic effect by enhancing the vascular protective ac-
                                                                  tions of the statin, and to prevent platelet
    Indeed, the administration of NCX 6550, but not of            hyperresponsiveness. The photochemical generation of
                                                                 al
pravastatin, strongly enhanced the levels in plasma of NO-        oxygen radicals in the femoral artery produced systemic
degradation products and of cGMP, confirming the in vivo          inflammation too, as shown by the rise of circulating IL-
                                                     ci


release of biologically relevant amounts of NO.                   6 and TNFα, which were significantly more reduced by
                                                   er



    Photochemical injury is a relatively non-invasive             NCX 6560 than by pravastatin.
method for inducing endothelial injury which is particu-              Statin therapy is associated with a small but significant
                                                  m




larly useful for small animals, such as mice.38 Endothelial       absolute increase in new onset diabetes, more marked in
                                         om




injury elicited by rose Bengal is mediated by the local           people with atherosclerosis and the metabolic syndrome.41
generation of superoxide anion,38 a type of injury that may       The diabetogenic risk appears to be lower with
                                                                  pravastatin.42 Pravastatin was reported to inhibit TNFα
                                    -c




                                                                  generation in vivo and indeed circulating TNFα levels of
                                                                  hypercholesterolemic patients were also significantly re-
                             on




                                                                  duced after 8 weeks of pravastatin treatment.43,44 Since
                                                                  TNFα plays a key role in the pathogenesis of insulin re-
                        N




                                                                  sistance and type 2 diabetes,45 the slow and sustained re-
                                                                  lease of NO from NCX 6550,46 producing a reduction of
                                                                  circulating TNFα (as we show here) may potentially con-
                                                                  tribute to the prevention of diabetes. Moreover, the release
                                                                  of NO in vivo in amount sufficient to inhibit the platelet
                                                                  hyperactivity induced by acute hyperglycemia in diabetic
                                                                  patients stimulates glucose transport in adipocytes without
                                                                  negatively affecting insulin sensitivity and it could there-
                                                                  fore help in better controlling glycemia in type 2 diabetes.47


Figure 6. Effect of pravastatin or NCX 6550 on (a) plasma         CONClUsIONs
NO2/NO3 production (n=7 mice per group), expressed as ng/ml
and (b) plasma levels of cGMP (n=5 mice per group), as meas-          In conclusion, our study shows that in a model of ac-
ured by ELISA in LDLR–/– mice fed an HFD. Statistical analy-      celerated atherosclerosis and increased thrombotic ten-
sis: ANOVA followed by the Newman-Keuls multiple                  dency a NO-donating pravastatin hybrid drug has a strong
comparisons test.                                                 and prompt antithrombotic, anti-inflammatory and anti-

                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:19
                  A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice                  13



atherosclerotic effect. The addition of a NO-donating mol-                    prevention: an analysis from the JUPITER trial. Lancet
ecule to pravastatin may enhance its effectiveness in dis-                    2012;380:565-71.
ease conditions of in vivo peroxidation and strong                      18.   Ongini E, Impagnatiello F, Bonazzi A, et al. Nitric oxide
atherosclerosis-related inflammation, such as those found                     (NO)-releasing statin derivatives, a class of drugs showing
                                                                              enhanced antiproliferative and antiinflammatory properties.
in high-risk patients with acute coronary syndromes, po-
                                                                              Proc Natl Acad Sci USA 2004;101:8497-502.
tentially reducing adverse events.                                      19.   Dever G, Spickett CM, Kennedy S, et al. The nitric oxide-
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                                                                              [1α(βS*,δS*),2α,6α,8β-(R*),(8aα]]-1,2,6,7,8,8a-Hexahydro
ReFeReNCes                                                                    β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy-1-
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                                          Bleeding, Thrombosis and Vascular Biology 2022; 1:19
