CMPAase and AREase activities in AIS
The first major finding of the present study is that increased AREase and reduced CMPAase activities are associated with AIS, and that these enzyme activities highly predict the degree of post-stroke impairments (mRS and NIHSS scores) at baseline and 3 and 6 months after AIS. Interestingly, both CMPAase and AREase activity further decreased from baseline to 3 months later.
PON1 is capable of hydrolyzing many lactones, arylesters, organophosphate insecticides, nerve gases, glucuronide medicines, and estrogen esters [41]. Using different substrates, the activity of PON1 can be evaluated to determine paraoxonase/phosphotriesterase activity (using paraoxon or CMPA as substrates), AREase activity (using phenylacetate or 4 (p) -nitrophenyl acetate as substrates), or lactonase activity (5-thiobutyl butyrolactone or dihydrocoumarin as substrates) [42].
Our work is the first to our knowledge to establish a correlation between PON1 CPMAase activity and AIS. Previous studies have shown a negative correlation between overall PON 1 activity and AIS [21, 43]. In some investigations, an inverse correlation between AREase serum activity and AIS was identified [44-45]. Some studies discovered that serum PON1 activity is connected with the functional prognosis of AIS patients and indicated that the mRS score of AIS patients tends to decrease as serum PON1 activity levels rise [21, 44]. On the other hand, Abdullah et al. [46] observed no association between serum PON1 activity and NIHSS in AIS patients. However, it is difficult to compare these findings to ours since earlier research overlooked the diverse enzyme activities of PON1. In this respect, CMPAase activity is also inversely associated with other neurodegenerative and neuroinflammatory disorders, such as deficit schizophrenia [40], temporal lobe epilepsy [47], and mood disorders, such as major depression and bipolar disorder [48], as well as the severity, progression, and neurocognitive impairments that accompany these disorders. Nevertheless, according to our findings, AIS is the only condition accompanied by concurrent increases in AREase and reductions in CMPAase activity.
Our findings that CMPAase (decreased) and AREase (increased) are differently associated with AIS and subsequent impairments demonstrate once again that the PON1 enzyme has (at least) two unique catalytic sites (CMPAase and AREase) [39], A newly estimated z unit-based composite score based on decreased CMPAase vs increased AREase activity was the best predictor of AIS and the mRS and NIHSS scores 3 and 6 months after acute stroke in our research. This demonstrates that decreased CMPAase coupled with elevated AREase are key contributors to AIS and subsequent disabilities. It appears that the PON1 enzyme has a Janus-face with two distinct catalytic sites that are differentially associated with AIS. As a result, we recommend assessing both CMPAase and AREase activities and computing their z-unit composite score as indicators of PON1 activity.
PON1 activities, HDL and AIS
The second major finding of our study is that CMPAase, but not AREase, activity was significantly correlated with HDL levels and that lowered levels of the newly calculated composite score of zCMPAase+zHDL (or combinations of low HDL with low CMPAase in GEE models) were the second-best predictors of AIS and its outcome. After its release in serum, PON1 binds to HDL thereby contributing to protective functions of HDL and preventing oxidative alterations of HDL and LDL, which are linked to several vascular disorders, including atherosclerosis [39, 49-50]. In fact, the PON1 enzyme accounts for the majority of HDL's antioxidant properties and shows anti-inflammatory, anti-apoptotic and antiatherogenic properties [39, 51].
Most AIS are thromboembolic in origin, originating from an atherosclerotic plaque [4], whilst reduced HDL and increased triglycerides and LDL are important biomarkers and risk factors of AIS because they are involved in the process of atherosclerosis [52, 54]. Several mechanisms have been proposed for the role of PON1 in the atheroprotective activity of HDL, including: a) making HDL more resistant to oxidation, thereby preserving its antiatherogenic capacity; b) inducing reverse cholesterol transport; c) decreasing and preventing oxidation of LDL by reducing the formation of foam cells; d) attenuated macrophage production of reactive oxygen radicals and inhibiting monocyte chemoattractant protein-1 (MCP) [22, 25, 55-56]. Moreover, the PON1-HDL complex has antioxidant, anti-inflammatory, antiapoptotic, vasorelaxant, and antithrombotic characteristics and supports endothelial function normalization and endothelial progenitor cell function stimulation [4, 39, 56].
The newly calculated composite score of zCMPAase+zHDL, therefore, represents the activity and functionality of the PON1-HDL complex in serum [57-58]. Consequently, our data revealing that this composite score is much lower in AIS and is inversely associated with post-stroke disabilities is in agreement with the knowledge that decreased antioxidant, anti-inflammatory, anti-apoptotic, and atherogenic protection is a crucial component of the pathophysiology of AIS. The loss of the above protective mechanisms (lowered CMPAase) may potentially lead to increased production of reactive oxygen species and activation of oxidative and inflammatory pathways, thereby interfering with the ability to protect HDL and LDL from oxidation [24-25, 39, 59].
It should be emphasized that serum CMPAase activity (but not AREase activity) was substantially linked with serum HDL concentrations in some neuropsychiatric illnesses (bipolar disorder and major depression) [57-58]. Moreover, mediation analysis demonstrated that the effects of CMPAase or the CMPAase-HDL complex on the phenome of these mood disorders, schizophrenia, and epilepsy are mediated by the effects of lipid peroxidation, including increased malondialdehyde formation [26, 58, 60]. These data may suggest that CMPAase in contrast to AREase is associated with increased oxidative stress and, as a result, exhibits higher antioxidant qualities than AREase, at least in neuropsychiatric disorders and AIS. Diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, liver and kidney disorders, including renal failure, psoriasis, and macular degeneration, are other diseases with a significant inflammatory and /or oxidative component that have been linked to dysfunctional PON1 and/or HDL [39, 61]. Future study should explore the two catalytic PON1 sites in those disorders and delineate the differences in substrates and properties of these different sites.
Inflammation, hypertension and PON1 activities in AIS
The third major finding of our study is that CRP influenced CMPAase (but not AREase) activity, HDL, and the CMPAase-HDL association (see results of PLS analysis). A recent meta-analysis showed that increased baseline hsCRP is associated with increased risk of AIS [62]. Alfieri et al. [18] found that hsCRP is highly linked with AIS and consequent disabilities at baseline and 3 months later. Moreover, higher CRP levels are related with poor outcomes as measured by the NIHSS scale, stroke subtypes, and traditional risk factors [63] and are reported as independent predictors of 1-year mortality [64]. CRP is thus a significant inflammatory biomarker of AIS and post-stroke prognosis [65-68]. Low PON1 activity has been associated with elevated CRP levels, indicating a mechanistic connection between PON1 activity and inflammation and the development of atherosclerosis [69].
Our findings also show that these decreases in CMPAase activity and in the zCMPAase-zAREase score from baseline to three months later predicted increased disability scores from baseline to 3 and 6 months later. It is plausible that the lowered CMPAase and AREase activities from baseline to three months later are caused by the above inflammatory processes. Activated macrophages promote the release of peroxides, nitric oxide, and myeloperoxidase, which, when combined, may form peroxynitrite and hypochlorous acid [57]. The latter are toxic products that may damage PON1 in HDL, whilst myeloperoxidase may further oxidize PON1, inactivating PON1 and reducing its binding to HDL [28]. Moreover, during systemic inflammation, native HDL may be transformed to dysfunctional HDL owing to the enzyme PON1's loss of antiatherogenic capabilities, which leads to the less protective and more atherogenic character of HDL [70].
Our investigation also revealed that the effects of hypertension on AIS are partly mediated by the effects of CRP on CMPAase and the CMPAase-HDL complex. The most frequently reported risk factor for AIS is hypertension [6, 71-72]. Inflammation, oxidative stress, and endothelial dysfunction have been linked to both hypertension and AIS pathogenesis [73]. Low PON-1 activity in the circulation has been observed in hypertensive individuals [74-75] and HDL may protect against hypertension due to its antithrombotic, antioxidant and anti-inflammatory effects, which reduce damage to the blood vasculature. HDL is also antiatherogenic due to its well-known role in reverse cholesterol transport, which modulates endothelial function in a favorable manner [76]. In addition, the underlying mechanisms through which HDL may reduce the incidence of hypertension comprise antioxidant and anti-inflammatory capabilities, which in fact are due to its PON1 individuals [77-78].
PON1 genotypes and AIS
Although there was no direct association between PON1 Q192R genotypes and AIS, the influence of PON1 genotypes on stroke became evident when multiple regression, GEE and PLS analyses were conducted. These analyses demonstrated a protective impact of QQ on NIHSS scores 3 and 6 months after baseline and that the RR genotype was linked with elevated mRS values 3 and 6 months after AIS. Liu et al. [79] conducted a comprehensive review and meta-analysis of AIS and PON1 gene polymorphisms and determined that the R allele or RR genotype of the PON1 Q192R polymorphism was associated with an elevated risk of AIS in the general population [79]. The homozygous QQ genotype may be protective against AIS in a Turkish population [80]. Nonetheless, not all investigations have confirmed these results [81-82]. One possibility is that the distribution of the PON1 genotype varies greatly across ethnic groups [30, 83-84] and that the altered PON1 genetic variations among different ethnicities may influence their vulnerability to AIS [25]. The benefits of the PON1 Q192R genotype on AIS have been attributed to the Q isoform inhibiting LDL oxidation more effectively than the R isoform [28, 85]. In addition, the R isoenzyme has less ability to hydrolyze lipid peroxide and, thus, poorer anti-atherogenic properties than the 192Q isoenzyme [86, 87]. Nevertheless, the R form has certain benefits since it can hydrolyze paraoxon more quickly than the Q type [88].
Previous research showed that patients with the PON1 QQ genotype demonstrate reduced PON1 activity at baseline and 12 months after a stroke compared to those with the RQ/RR genotypes (Shenhar-Tsarfaty et al., 2013). However, our data also indicate that the PON1 QQ genotype, which may have protective effects against AIS, substantially decreases CMPAase and increases AREase activity, which both increase risk to AIS. Furthermore, our PLS analysis revealed that the associations between the PON1 Q192R genotype, the PON1 activities and AIS are far more complex than can be estimated from association studies or regression analysis. Thus, our PLS analysis demonstrates that a) the RR genotype is associated with increased AIS risk via a direct pathway, b) the QQ genotype is associated with increased AIS risk via two indirect pathways, namely via CMPAase (and the CMPAase-HDL complex) and AREase, which both yield a negative direction despite the fact that the effects of the genotype on both enzymatic activities are completely opposite; and c) the direct and indirect paths have opposite effects leading to an overall effects that is not significant. Thus, the PON1 gene has complex, multiple Janus-faced effects on AIS outcome which can only be delineated using mediation analysis and including both catalytic enzyme activities. This view is supported by our neural network results which revealed that the Q192R genotype and both PON1 activities contribute considerably to the prediction of AIS.