antioxidants Review Therapeutic Potential of Emerging NAD+-Increasing Strategies for Cardiovascular Diseases Noemi Rotllan 1,2,3,† , Mercedes Camacho 1,4,† , Mireia Tondo 2,3,5 , Elena M. G. Diarte-Añazco 1 , Marina Canyelles 1,2,3 , Karen Alejandra Méndez-Lara 1 , Sonia Benitez 1 , Núria Alonso 3,6 , Didac Mauricio 3,7 , Joan Carles Escolà-Gil 1,2,3, * , Francisco Blanco-Vaca 2,3,5, * and Josep Julve 1,2,3, * 1 Institut de Recerca i d’Investigació Biomèdica de l’Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, 08041 Barcelona, Spain; nrotllanv@santpau.cat (N.R.); mcamacho@santpau.cat (M.C.); ediarte@santpau.cat (E.M.G.D.-A.); mcanyelles@santpau.cat (M.C.); kmendez@santpau.cat (K.A.M.-L.); sbenitez@santpau.cat (S.B.) 2 Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; mtondo@santpau.cat 3 CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, 28029 Madrid, Spain; nalonso.germanstrias@gencat.cat (N.A.); dmauricio@santpau.cat (D.M.) 4 CIBER de Enfermedades Cardiovasculares, CIBERCV, 28029 Madrid, Spain 5 Department of Biochemistry, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, 08041 Barcelona, Spain 6 Department of Endocrinology & Nutrition, Hospital Universitari Germans Trias i Pujol, 08916 Barcelona, Spain 7 Department of Endocrinology & Nutrition, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, 08041 Barcelona, Spain * Correspondence: jescola@santpau.cat (J.C.E.-G.); fblancova@santpau.cat (F.B.-V.); jjulve@santpau.cat (J.J.);   Tel.: +34-935537588 (J.C.E.-G.); +34-935537377 (F.B.-V.); +34-935537586 (J.J.) † First coauthors. Citation: Rotllan, N.; Camacho, M.; Tondo, M.; Diarte-Añazco, E.M.G.; Abstract: Cardiovascular diseases are the leading cause of death worldwide. Aging and/or metabolic Canyelles, M.; Méndez-Lara, K.A.; stress directly impact the cardiovascular system. Over the last few years, the contributions of altered Benitez, S.; Alonso, N.; Mauricio, D.; nicotinamide adenine dinucleotide (NAD+) metabolism to aging and other pathological conditions Escolà-Gil, J.C.; et al. Therapeutic Potential of Emerging NAD+- closely related to cardiovascular diseases have been intensively investigated. NAD+ bioavailability Increasing Strategies for Cardiovascular decreases with age and cardiometabolic conditions in several mammalian tissues. Compelling data Diseases. Antioxidants 2021, 10, 1939. suggest that declining tissue NAD+ is commonly related to mitochondrial dysfunction and might https://doi.org/10.3390/antiox10121939 be considered as a therapeutic target. Thus, NAD+ replenishment by either genetic or natural dietary NAD+-increasing strategies has been recently demonstrated to be effective for improving the Academic Editor: Stanley Omaye pathophysiology of cardiac and vascular health in different experimental models, as well as human health, to a lesser extent. Here, we review and discuss recent experimental evidence illustrating that Received: 26 October 2021 increasing NAD+ bioavailability, particularly by the use of natural NAD+ precursors, may offer hope Accepted: 27 November 2021 for new therapeutic strategies to prevent and treat cardiovascular diseases. Published: 3 December 2021 Keywords: macrophage; aneurysm; atherosclerosis; ischemia/reperfusion; cardiomyopathy; heart Publisher’s Note: MDPI stays neutral failure; chemotherapy; clinical trials; COVID-19; niacinamide; vitamin B3; niacin; niagen; niaspan; with regard to jurisdictional claims in mitochondria; tryptophan; diabetes; myocarditis; animal models; clinical trials published maps and institutional affil- iations. 1. Introduction Copyright: © 2021 by the authors. Cardiovascular diseases are the leading cause of death worldwide according to the Licensee MDPI, Basel, Switzerland. World Health Organization. Aging is a primary risk factor for many cardiovascular diseases. This article is an open access article With the progressively aging population, the elevated prevalence of cardiovascular diseases distributed under the terms and undermines human health and longevity [1]. conditions of the Creative Commons Mitochondrial dysfunction is a hallmark of metabolic decline during aging [2]. Nicoti- Attribution (CC BY) license (https:// namide adenine dinucleotide (NAD+) bioavailability decreases with age in a number of creativecommons.org/licenses/by/ mammalian tissues, and evidence suggests that failing mitochondrial function is related to 4.0/). this decline [3,4]. Beyond aging, altered NAD+ metabolism levels have also been reported Antioxidants 2021, 10, 1939. https://doi.org/10.3390/antiox10121939 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1939 2 of 41 in obesity, diabetes mellitus, fatty liver disease, and hypertension [5]. NAD+ depletion is observed in several cardiovascular pathologies, including atherosclerosis, ischemia- reperfusion injury, cardiomyopathy, and heart failure (HF), which has prompted studies exploring its pathophysiological role in cardiovascular diseases [6]. In this regard, NAD+ decline prevention may represent an innovative therapeutic target in the management of cardiovascular diseases [7]. Accordingly, many studies have revealed that dietary adminis- tration of NAD+ precursors, such as tryptophan, nicotinic acid (NA, also termed niacin), nicotinamide (NAM), and nicotinamide riboside (NR), and its phosphorylated form, nicoti- namide mononucleotide (NMN), efficiently raises the tissue NAD+ content in rodents and humans [8,9]. In particular, NAM, NMN, and NR administration has been reported to prevent obesity and improve glucose tolerance in mice [10–14] and favorably influenced fatty liver by reducing oxidative stress and inflammation in aged, diet-induced obese mice [15]. Conceivably, NAD+ precursors may protect the cardiovascular system [6,16]. NA is a vitamin B3 form that has been widely used in clinical practice to reduce the atherothrombotic cardiovascular risk by decreasing plasma triglycerides and increasing plasma high-density lipoprotein (HDL) cholesterol (HDL-C) [17]. NA interacts with the GPR109A receptor in immune cells, thereby blunting immune activation and protecting against inflammatory cell infiltration in age-related diseases [18]. Recent studies suggest that many physiological effects of NA on cardiovascular protection can be at least partly explained on the basis of its role as an NAD+ precursor [19]. However, a major problem with NA administration is adverse effects, which range from cutaneous flushing to effects linked to muscle, liver, and gastrointestinal toxicity [20]. Moreover, early reports supporting secondary cardiovascular preventive effects of NA were not confirmed by more recent, large prospective trials including 29,087 patients, the AIM-HIGH [21] and the HPS2-THRIVE [22], which were prematurely stopped due to a lack of efficacy in patients that were taking statins in the context of secondary prevention. Thus, these results do not, therefore, necessarily apply to primary cardiovascular prevention, but not such type of novel NA clinical trial seems to be planned at this point. In a recent study [23], NA also ameliorated wound healing and cardiac function after myocardial infarction via DP1-mediated M2 macrophage polarization and timely resolution of inflammation, thus suggesting that DP1 inhibition, as the agent used together with NA in the abovementioned large clinical trials, may attenuate the cardiovascular benefits of NA. The potential benefits of other forms of vitamin B3 have also been studied, albeit more experimentally than at the clinical level. NAM, an amide vitamin B3 form, does not interact with the GPR109A receptor and is apparently free of NA-induced adverse side effects [7,8]. Treatment with both NAM and NA improved aneurysms in an experimental mouse model through GPR109A receptor-independent activity against this disease [24]. Unlike NA, NAM does not show strong lipid-lowering effects [19] but exerts antioxidant and anti-inflammatory effects [15,25]. Interestingly, NAM administration protected against beta pancreatic cell damage and loss and, thus, delayed the onset of diabetes mellitus type 1 in experimental models [26]. Although this effect was not reproduced in the ENDIT clinical trial [27], NAM administration at a dose of 1.2 g/m2 did not cause adverse effects (including flushing) similar to those attributed to NA. Recent data from our own group show that NAM prevents inflammation and atherosclerosis in experimental animals [25]. Similarly, some other related compounds, such as NR [28], NMN [9], and N-methylated NAM (me-NAM) [29,30], seem to protect against vascular damage. Some evidence indicates that NAM or other related compounds that act as a source of NAD+ might be of interest in the context of cardiomyopathy and heart failure (HF) [31]. Thus, this comprehensive review provides updated, critically revised information on the potential therapeutic effects of boosting NAD+ metabolism in animal models of cardiovascular diseases and its potential translation into clinical practice. In particular, we mainly focus on the role of NAD+-increasing strategies based on the use of NAD+ precursors. Other determinants of NAD+ metabolism also lead to NAD+ elevations; however, owing to space limitations, genetically modified mouse models are not extensively Antioxidants 2021, 10, x 3 of 38 Antioxidants 2021, 10, 1939 mainly focus on the role of NAD+-increasing strategies based on the use of NAD+ precur- 3 of 41 sors. Other determinants of NAD+ metabolism also lead to NAD+ elevations; however, owing to space limitations, genetically modified mouse models are not extensively dis- cussed in this review. Furthermore, although various pharmacological strategies have discussed also in this review. been reported Furthermore, to boost although NAD+ levels, various including pharmacological the use strategies of activators and have inhibitors of also been reported to boost NAD+ levels, including the use of activators and NAD+ biosynthesis, we focus on the therapeutic potential of direct natural NAD+ precur- inhibitors of NAD+ sors biosynthesis, in cardiovascular we focus on the therapeutic potential of direct natural NAD+ health. precursors in cardiovascular health. 2. Preferential Physiological Sources of NAD+ 2. Preferential Physiological Sources of NAD+ NAD+ is an essential cofactor in all living cells [32]. Abundant amounts of this me- NAD+ is an essential cofactor in all living cells [32]. Abundant amounts of this metabo- tabolite are found in different cellular compartments, including mitochondria, cytoplasm, lite are found in different cellular compartments, including mitochondria, cytoplasm, and and nuclei. NAD+ plays a key physiological role in modulating many biological processes nuclei. NAD+ plays a key physiological role in modulating many biological processes and cellular metabolism pathways, including glycolysis, fatty acid β-oxidation, and the and cellular metabolism pathways, including glycolysis, fatty acid β-oxidation, and the tricarboxylic acid cycle [33,34]. NAD+ exists in two forms—the oxidized form (NAD+) and tricarboxylic acid cycle [33,34]. NAD+ exists in two forms—the oxidized form (NAD+) the reduced form (NADH). The latter is generated by NAD+ accepting high-energy elec- and the reduced form (NADH). The latter is generated by NAD+ accepting high-energy trons from glycolytic and tricarboxylic acid intermediates and acts as a primary electron electrons from glycolytic and tricarboxylic acid intermediates and acts as a primary electron donor in ATP production by feeding electrons into complex I of the electron transport donor in ATP production by feeding electrons into complex I of the electron transport chain chain to drive mitochondrial oxidative phosphorylation [35]. to drive mitochondrial oxidative phosphorylation [35]. 2.1. 2.1. NAD+ NAD+ Sources Sources Because NAD+-dependent signaling Because NAD+-dependent signalingreactions reactions(protein (proteindeacetylation deacetylation byby sirtuins, sirtuins, in- intra- tracellular calcium signaling, and mono-/poly-ADP-ribosylation) involve cellular calcium signaling, and mono-/poly-ADP-ribosylation) involve degradation of the degradation of the dinucleotide, a constant supply of this molecule is required to reach cellular dinucleotide, a constant supply of this molecule is required to reach cellular homeostasis. homeo- stasis. NAD+NAD+ precursors precursors are commonly are commonly ingestedingested from from dietary dietary sources. sources. Intracellular Intracellular NAD+ NAD+ can can be technically be technically produced produced through through distinct distinct metabolic metabolic pathways pathways (Figure (Figure 1): (i)1): the(i)dethe novode novo pathway pathway from from tryptophan, tryptophan, (ii) the(ii)salvage the salvage synthesis synthesis pathway pathway fromfrom NAMNAM and NR,and and NR, and (iii)Preiss-Handler (iii) the the Preiss-Handler pathway pathway fromfrom NA [32,36]. NA [32,36]. Additionally, Additionally, intermediate intermediate mole- molecules cules from from these these NAD+ NAD+ biosynthetic biosynthetic pathways, pathways, suchsuch as NMN, as NMN, can also can also be direct be direct substrates substrates for for NAD+ NAD+ synthesis synthesis [33].[33]. Tryptophan IDO TDO Kynurenine KMO KYNU 3HAO De novo pathway Quinolinic acid + NAD consuming enzymes NR Nrk1/2 NMN Nnmat NAD+ PARPs Sirtuins cADPr synthases Nmat Preiss- SARM1 Handler Nampt Nprt pathway Salvage pathway NA NAM Nnmt me-NAM Figure 1. NAD+ precursor metabolism metabolismand andNAD+-consuming NAD+-consumingenzymes. enzymes.Tryptophan, Tryptophan,NA,NA, NAM, NAM, and and NR be NR can canused be used by different by different pathways pathways to obtain to obtain NAD+. NAD+. NAD+NAD+ formation formation can can be obtained be obtained de de novo novo from tryptophan. from tryptophan. The The first first step stepinrelies relies in the conversion the conversion of tryptophan of tryptophan to N- to N-formylkynurenine by either indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). N-formylkynurenine can be converted into quinolinic acid and NAD+ thereafter. NAD+ synthesis from NA is initiated by the NA phosphoribosyltransferase (NPRT), which uses phosphoribosyl pyrophosphate (PRPP) to form NAMN prior to NAD+ synthesis (Preiss-Handler pathway). Finally, the synthesis of NAD+ can be Antioxidants 2021, 10, 1939 4 of 41 also possible using NAM or NR via the Salvage pathway. This pathway just needs 2 steps for each precursor. NAM is converted by the rate-limiting nicotinamide phosphoribosyltransferase (NAMPT) to form NMN, also using PRPP as a co-substrate. NMN can be also formed by NR phosphorylation through the action of NR kinases (NRK1-2). Finally, the conversion of NMN to NAD+ is eventu- ally catalyzed by cellular NMN adenylyl transferase (NMNAT1-3) enzymes. Abbreviations used were: 3HAO: 3-hydroxyanthranilate 3,4-dioxygenase; IDO: indoleamine 2,3-dioxygenase; KMO: kynureine monooxygenase; KYNU: kynureinase; NAD: nicotinamide adenine dinucleotide; NA: nicotinic acid; NAM: nicotinamide; NMN: nicotinamide mononucleotide; Nampt: nicotinamide phosphoribosyltransferase; Nnmat: nicotinamide mononucleotide adenylyltransferase; NR: nicoti- namide riboside; Nrk1/2: NR kinase 1, 2; Nmat: nicotinate mononucleotide adenyltransferase; Nprt: nicotinate phosphoribosyl-transferase; Nnmt: nicotinamide methyltransferase; TDO: tryptophan 2,3-dioxygenase. 2.1.1. De Novo Synthesis Pathway The kynurenine pathway drives de novo synthesis of NAD+ from the essential amino acid tryptophan [37] (Figure 1). This metabolic pathway is the dominant route of metabolism for absorbed tryptophan [38], accounting for more than 95% of daily me- tabolized tryptophan. However, tryptophan is considered an inefficient source of NAD+, as it requires 8 enzymatic reactions and, thus, has lower therapeutic value than other precursors, including NAM, NR, and NMN [39]. Therefore, these last molecules can be considered much more physiologically preferred NAD+ precursors than tryptophan [40]. Although the potential function of the kynurenine pathway of tryptophan metabolism to produce NAD+ has received little attention, emerging evidence suggests potential interplay between this pathway and cardiovascular disease [37,41]. In line with this, the kynurenine pathway has been recently regarded as a potential source of NAD+ in macrophages [42]. 2.1.2. Salvage Pathway NMN is a major intermediate of NAD+ (Figure 1). The salvage synthesis of NMN from NAM is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT), whereas the synthesis of NMN from NR is catalyzed by intracellular NR kinases (NRK1 and NRK2) [43]. Finally, NMN may be subsequently transformed into NAD+ by different subcellular forms of adenylyltransferases (NMNAT1-3) [44]. 2.1.3. Other Modulators of Intracellular NAD+ Pools In addition to these enzymes, others may also influence the tissue abundance of NAD+ (Figure 1). Specifically, nicotinamide N-methyltransferase (NNMT) catalyzes the N- methylation reaction of NAM and other analogous molecules using S-adenosyl methionine (SAM) as a donor of methyl groups [45] and produces me-NAM and S-adenosyl homocys- teine (SAH) [39,40]. Recent reports suggest that NNMT may also act as a determinant of tissue NAD+ pools in vivo [43]. Interestingly, the subproduct me-NAM has been reported to exert anti-inflammatory, antioxidant, and antidiabetic effects in different experimental models [46–50]. 2.2. Administration and Bioavailability of NAD+ Precursors L-tryptophan can be safely supplemented in the diet [51]. This amino acid can be added directly to feedstuff and dissolved into drinking water [38]. As indicated previously, its availability is primarily governed by the kynurenine pathway, which may contribute to NAM production under dietary deficiency of this vitamin form [38]. NAM is highly soluble in water and is considered a safe food supplement in animal nutrition and the predominant circulating form of vitamin B3 in vivo [52]. Its safety margin is at least ten times the dietary requirements and use levels, and it can be administered orally by feeding or drinking (tap water) routes with similar bioequivalence rates. Oral NAM can be absorbed in the stomach and the small intestine either through passive diffusion or Antioxidants 2021, 10, 1939 5 of 41 by Na+-dependent carrier-mediated facilitated transport mechanisms [52], with passive diffusion predominating at pharmacological doses. Similarly, NA absorption into the intestinal mucosa follows the same transport mechanisms as NAM. NA can be further metabolized via NA mononucleotide (NAMN) to form the NA analog of NAD+ through the Preiss-Handler pathway (Figure 1). NAD+ can then be enzymatically cleaved into NAM in the intestinal mucosa and then the liver. The latter tissues contain elevated amounts of NAD+-consuming enzymes, such as NAD+ glycohydrolase, that allow NAM synthesis from NAD+. NAM can be subsequently transported to tissues for the synthesis of NAD+ or its phosphorylated form (NADP+). The principal excretory product is the methylated, water-soluble metabolite me-NAM (Subsection: Other modulators of intracellular NAD+ pools) or some of its oxidation products [53]. NR has a good bioavailability [54]. Unlike NA and NAM, dietary NR cell uptake is mediated by a specific nucleoside transporter [55], where it is transformed into NAD+ through a distinct, two-step biosynthetic pathway. In contrast to that of NR, the bioavailability of NMN is rather limited [56]. Indeed, extracellular sources of NMN do not efficiently cross the endothelial membrane and require previous dephosphorylation by CD73 (ecto-50 -nucleotidase) to generate NR or must be extracellularly metabolized to NAM by CD38 [57]. Interestingly, a specific NMN transporter (i.e., Slc12a8) exists in murine small intestinal epithelium [58]. This transporter is predominantly expressed in the small intestine and in white adipose tissue and the liver to a lesser extent. However, whether Slc12a8 can also be expressed by cells of the vasculature or cardiomyocytes remains to be determined. NMN can also be obtained from NAM by extracellular NAMPT (eNAMPT, also termed visfatin). In endothelial cells, this conversion process requires the participation of phosphoribosyl-1-pyrophosphate (PRPP) [8]. Finally, extracellular NAD+ can be directly transported into cardiomyocytes through connexin 43 (i.e., Cx43) channels [59]. Taken together, these findings suggest that intracellular transport of NAD+ and NMN may be cell-specific and rely on different transporters. 2.3. NAD+-Consuming Enzymes as Determinants of the NAD+ Content In the last two decades, beyond its important contribution to intermediate metabolism, NAD+ has also been shown to influence multiple signaling pathways (reviewed in detail in ref. [8]). Indeed, NAD+ can also be used by different protein regulators, including NAD+-dependent histone deacetylases (also termed sirtuins), which regulate the cellular mitohormetic response and, thus, favorably influence energy metabolism (reviewed in detail in ref. [33]). Moreover, several families of ADP-ribose synthesizing enzymes—mono- (ADP-ribose) polymerases (MARPs) and poly-(ADP-ribose) polymerases (PARPs)—also require NAD+ to specifically catalyze mono-/poly-ADP-ribosylation reactions of target proteins [60]. These enzymes are generally considered DNA damage sensors because of their participation in DNA repair, replication and transcription and the chromatin structure, among other functions [33,34,61–64]. Other enzymes, such as NAD+-dependent glycohy- drolases (i.e., CD38 and CD157) involved in calcium signaling [65,66] and regulation of the cell cycle [66–68], use NAD+ to produce the second messenger cyclic-ADP-ribose and ADP-ribosyl cyclase-produced glycoproteins [69]. 3. In Vitro Cardioprotective Effects of NAD+ Sources Experimental evidence suggests that oxidative stress and inflammation are mechanis- tically interconnected in driving cardiac and vascular diseases, and both may contribute to disease progression [70]. Increased vascular reactive oxygen species can react with cellular molecules and organelles, which become dysfunctional, leading to inflammation and endothelial dysfunction with devastating consequences on the vasculature and cardiac tissue. Moreover, impaired vascular reactive oxygen species homeostasis and inflammation may be reciprocally exacerbated, thereby resulting in a vicious and chronic cycle. Antioxidants 2021, 10, 1939 6 of 41 Current applications for successful control of enhanced oxidative stress and inflamma- tion for the management of such complex chronic diseases are still elusive, partly due to the extensive diversity of oxidative stress pathways and inflammatory cells that can either be cardioprotective or cause cardiac damage. In this context, we next focus on relevant recent research evidence to examine some cardioprotective properties shown by vitamin B3 forms or intermediates in appropriate cell models of disease. 3.1. Ex Vivo Properties of NAD+ Precursors Relevant to Cardiovascular Diseases Reactive oxygen species, including the superoxide anion (O2 •− ) and the nitric oxide radical (NO• ), are critical bioreactive signaling molecules that may influence both cellular homeostasis and disease. Under physiological conditions, the intracellular pool of such reactive oxygen species is controlled by an intricate array of antioxidant defense systems that include superoxide dismutases, catalase, the glutathione peroxidase/reductase system, and the peroxiredoxin/thioredoxin system [71]. Under pathological conditions, elevated reactive oxygen species can overwhelm cellular antioxidant defenses, impair reactive oxygen species scavenging and, therefore, lead to cardiac dysfunction [72–76]. In addition to the enzymatic antioxidant systems, certain molecules (identified as antioxidants) may serve as direct metabolic scavengers of electrons from reactive radicals and, thus, confer direct protection to biomolecules from oxidative damage by virtue of their chemical ability to accept/donate electrons. In particular, NAM may directly func- tion as an O2 •− radical scavenger and inhibit the generation of free radicals (e.g., O2 •− , NO• , and HCLO• ) [77] and prevent protein and lipid oxidation in vitro [78]. Importantly, this scavenging ability may paradoxically enable some vitamins with recognized antioxi- dant properties to function as pro-oxidants under certain circumstances [79–81]. Notably and in contrast to other vitamins with antioxidant abilities, such as vitamins A and C, vitamin B3 (NA) did not induce oxidative damage in vitro at physiologically relevant concentrations [82]. Both the resistance to oxidation of non-HDL and the antioxidant properties of HDL are two of the most important antiatherogenic metrics of lipoproteins ex vivo [83]. We recently reported that isolated plasma non-HDL from mice lacking apolipoprotein (Apo)E (Apoe−/− ) treated with NAM was less susceptible to oxidation ex vivo than that from untreated mice [25]. Notably, incubation of human low-density lipoprotein (LDL) with NAM produced a direct inhibitory effect on lipid peroxidation during an analysis of the oxidation kinetics of exposed lipoproteins [25]. However, the effect of NAM on the HDL antioxidant capacity of Apoe−/− mice could not be evaluated in the latter study due to the limited circulating HDL moieties in this mouse model. Instead, the effect was directly assessed using HDL isolated from wild-type mice on the same genetic background as the Apoe−/− mice. Consistently, our data revealed that HDL isolated from the plasma of NAM-treated mice was more protective against LDL oxidation ex vivo than HDL isolated from untreated mice (Julve et al., 2021, unpublished data, Figure 2), suggesting enhanced atheroprotection by HDL from NAM-treated mice. Antioxidants 2021, 10, x 7 of 38 Antioxidants 2021, 10, 1939 7 of 41 Figure 2. Effect of NAM manipulation on antioxidant ex vivo protection conferred by plasma isolated FigureHDL. 2. Effect of NAM manipulation (a) Representative on antioxidant diene formation curves ofexhuman vivo protection conferred LDL incubated withby plasma mouse HDL iso-isolated lated HDL. (a) Representative diene formation curves of human LDL incubated from untreated and NAM-treated wildtype C57BL/6J mice in the presence of 2.5 µM CuSO4 at with mouse HDL isolated 37 from untreated ◦ C. The and NAM-treated final oxidation wildtype kinetics of the LDL+HDL C57BL/6J mice mixture wasinshown the presence of 2.5 μM the kinetics after subtracting CuSO4 at 37 °C. The final oxidation kinetics of the LDL+HDL mixture was shown after subtracting of HDL incubated without LDL. (b) HDL antioxidant activity against LDL oxidative modification. the kinetics of HDL incubated without LDL. (b) HDL antioxidant activity against LDL oxidative Methods used can be found in greater detail in reference [84]. Data were expressed as relative lag modification. Methods used can be found in greater detail in reference [84]. Data were expressed phase to LDL oxidized in the presence of HDL isolated from Untreated mice (arbitrary unit = 1). as relative lag phase to LDL oxidized in the presence of HDL isolated from Untreated mice (arbi- Data=were trary unit expressed 1). Data were expressed as the ± as the median interquartile median range (n ± interquartile = 4–5(nindependent range pools per = 4–5 independent poolsgroup). per group). Differences between the mean values were determined using a nonparametric Mann– test. Differences between the mean values were determined using a nonparametric Mann–Whitney * indicates Whitney significant test. * indicates differences significant versus untreated differences mice (p