*5.1. Heart Failure*

JAK/STAT pathway transmits not only inflammatory signals, but also is deeply involved in the proper functioning of many systems of the body including the cardiovascular system. In neonatal rat, cardiocytes angiotensin II induces JAK2 phosphorylation and this process is critically depended on reactive oxygen species generation via membrane-bound NADP-oxidase. This may offer an important link between high glucose levels and glucose-dependent angiotensin II-mediated phosphorylation of JAK2. Of note is the fact that cardiac hypertrophy in non-failing heart is dependent on JAK2 phosphorylation [58]. This may potentially offer therapeutic approach in patients with RA, where insulin resistance and latent diabetes contribute to cardiac hypertrophy and heart failure. On the other hand, it was shown in a mice model that gp130 receptor and gp130 cytokines may offer survival pathway in transition to the heart failure (Figure 1) [59]. Thus, switching off this pathway via JAK inhibition ameliorates compensatory effect upon heart at risk for even failure. The pivotal role of cytokine that utilized gp130 receptor, namely IL-6 is still the matter of controversy. Contrary to TNF, which is known to contribute to heart insufficiency, the role of IL-6 is widely unknown. Quite recently, Hengdong et al. in their meta-analysis showed increased level of IL-6 was independently associated with higher risk of major adverse cardiovascular events (MACE), cardiovascular and all-causes of mortality in patients with acute coronary syndromes [60]. Although at this moment, it is not clear if IL-6 is the only valuable biomarker of heart failure or it is only pathophysiologically involved in heart failure. More information was obtained from the study of Liangjie et al. who assayed IL-6 and IL-17 in patients who underwent cardiac catherization. In this study, IL-6 and IL-17 levels were correlated with the levels of fibrotic parameters indicating the role of both cytokines in the development of heart insufficiency [61]. Recently, some indirect data suggested that targeting IL-1 with subsequent reduction of IL-6 exerted a significant effect on the primary cardiovascular end point [62]. IL-6 is the central inflammatory cytokine, and together with its downstream inflammatory biomarker CRP is linked to high cardiovascular risk [63].

Despite numerous experimental and clinical studies, the role of IL-6 on the development of heart failure has not yet been fully elucidated. It is believed that high concentration of IL-6 can lead at least partially to the development of heart failure [64] and may serve as an indicator of worse prognosis in patients with cardiovascular diseases [65]. Targeting JAK with subsequent blockade of gp130 mediated pathway may potentially facilitate to stabilize the heart function. At this moment, it is unclear whether this improvement is due to the direct reduction of IL-6 or this process is mediated via limitation of an inflammatory state. Blocking the JAK/STAT pathway may also bring many negative consequences. It is well known that activation of the JAK2/STAT3 pathway protects the myocardium against ischemia/reperfusion injury and inhibits apoptosis of the coronary artery endothelial cells [66], thus provide mechanism that are far beyond only cytokine signaling [67]. Contrary to this, in another study, inhibition of JAK2/STAT partially attenuated the pro-apoptotic effect of IL-23 (a member of

IL-12 family) upon cardiomyocytes [68]. Therefore, it is suggested that, in these circumstances, IL-23 promotes the activation of JAK2/STAT pathway and enhances the expression of IL-17, the cytokine deeply involved in myocardial ischemia/reperfusion injury (Figure 1) [69]. It is an identical fact when the other member from this cytokine family, IL-12, is concerned.

**Figure 1.** Several cytokine families utilize type I receptors. Receptors with gp130 component transmits signals from IL-6, IL-11, and IL-27 with subsequent activation of JAK1-JAK2 and TYK2 molecules. Cytokines activate (mainly IL-6) heart survival pathway resulting in stabilization of the heart function in ischemia/reperfusion conditions. The same pathway contributes however in deterioration of heart function and increases the risk of major adverse cardiovascular events (MACE), leading to heart fibrosis with subsequent development of heart failure. Cytokines IL-12 and IL-23 that interact with p40 receptor component transmit signals via activation of JAK2 and TYK2 molecules resulting in cardiomyocytes apoptosis. Moreover IL-12 facilitates IL-17 overexpression leading to myocardial/reperfusion injury. Blocking JAK/STAT pathway with JAK inhibitors may therefore result in blocking the heart failure survival pathway but also may reduce MACE incidence and fibrosis of the heart. Blocking of JAK/STAT pathway (mainly IL-6 mediated arm) is also responsible for unfavorable lipids profile changes mediated by reduced LDL catabolism, but this effect may be ameliorated by reduced expression of scavenger receptor class B and ATP-binding cassette G-1. Leading to improvement of lipid composition. Several pathological conditions upregulate JAK/STAT pathway activity (hyperglycemia, reactive oxygen species formation, and angiotensin II) facilitating transmission signals from proinflammatory cytokines.

Previous studies reported that plasma IL-12 concentrations were significantly increased in many types of atherosclerosis and atherosclerotic cardiovascular disease. At this moment, it is however unclear whether inhibition of signaling pathways via JAK/STAT system may attenuate the harmful effect of IL-12 upon the heart, which is not a surprising finding. The inhibition of JAK results in the reduced expression of many cytokines belonging to the various families. Moreover, even in the same cytokine family, some of them exert pro-inflammatory response since the other cytokines are recognized as anti-inflammatory ones. IL12 and IL-23 demonstrate strong pro-inflammatory properties. However, the other member of this family, IL-35, exerts anti-inflammatory potentials. Therefore, the direct effect

of the inhibition of the JAK/STAT pathway is dependent on what cytokines are predominantly blocked when inhibition of the JAK/STAT pathway occurs (Figure 2).

**Figure 2.** JAK inhibitors targeting JAKs of type I and II receptors. Based on cytokine profile inhibition of JAK/STAT pathway, diverse biological consequences are observed. Inhibition of JAK attached to γ-chain receptor resulting either in beneficial (blocking IL-15, high concentration mediated IL-2 transmission) or detrimental (inhibition of beneficial activity of low IL-2-impaired tissue healing and repair). Inhibition of JAK fused with gp130 receptor reduces IL-6 level. Based on the pathophysiological circumstances, reduced level of IL-6 may contribute to the reduction of heart survival pathway activity or favorably modify heart failure pathway. As far as IL-12 operating via p40 receptor subunit is concerned, inhibition of JAK results in reduction of IL-12-mediated signaling and exerts favorable effects on the cardiovascular system halting progression of atherosclerosis, reducing risk of developing ischemic cardiomyopathy, and myocardial fibrosis. Inhibition of JAK/STAT system transmitting signal from interferon receptor results in reduction of activity of IFN-dependent genes that translates directly to the reduction of foam cell formation and halting progression of atherosclerosis. Finally, some negative consequences may arise as the result of erythropoietin blockade with subsequent anemia development (indirectly contributing to worsening of heart function).

More data were provided from clinical studies where cardiovascular risk was assessed. Those studies demonstrated a low-incidence rate of MACE in RA patients, suggesting a good cardiovascular profile of JAK inhibitors [70]. Recently, these findings were substantiated by the first meta-analysis exploring the relationship between JAK inhibitor treatments and cardiovascular risks. According to the data from this study, short-term treatment with JAK inhibitor does not increase the risk of cardiovascular events when compared to placebo. Furthermore, with the exception of baricitinib, tofacitinib in both 5 and 10 mg doses and upadacitinib (15 mg and 30 mg doses) appeared to be equally safe [71]. Same conclusions were observed from analysis of clinical trials with tofacitinib [72]. The post-hoc analysis comprised in total eight trails - six phase III and long-term extension, respectively. The authors focused on MACE defined as any myocardial infarction, cerebrovascular event (i.e., stroke), or cardiovascular death (defined as death caused by coronary, cerebrovascular, or cardiac events) incidence in a large cohort of 4076 patients representing a total of 12,932 patient-years of tofacitinib exposure. In the study, MACE incidences were linked with older age, longer disease duration, higher mean body mass index, diabetes mellitus, hypertension, and lipid profile changes (higher total low-density lipoprotein (LDL), lower high-density lipoprotein (HDL) cholesterol, triglycerides,

and higher total cholesterol to LDL ratio). Contrary to the previous studies linking disease activity with increased risk of poor cardiovascular outcome, in this study, disease activity parameters and inflammatory measured as baseline disease activity and inflammation measures were not significantly associated with MACE [72].

#### *5.2. Lipid Profile*

RA is linked with increased risk of cardiovascular events. In general population, the role-playing risk factors for atherosclerosis and poor cardiovascular outcome are obesity, sedentary lifestyle, smoking, and lipid profile disturbances. However, a significantly higher risk of developing cardiovascular events in RA patients cannot be explained by the presence of traditional risk factors alone. For many years, RA and other inflammatory conditions are recognized as the independent risk factors. In line with this, hampering the disease activity may add extra beneficial effect on the cardiovascular system.

Patients with RA often show lipid paradox, having lower total cholesterol and its subfractions as compared to unaffected population [73,74]. The most common explanation of this phenomena is suppression of cholesterol synthesis by inflammatory processes. Indeed, there is a strong link between C-reactive protein (a biomarker of inflammation) and circulating lipid levels [74]. Furthermore, treatment of RA may also impact lipid profile and some DMARDs exert the potentials to increase serum LDL and HDL cholesterol levels [75]. These phenomena cannot be explained only by the reduction of inflammation, since impact of various DMARDs on the lipid profile is variable, in spite of similar reductions in disease activity and systemic inflammatory parameters [76,77]. Followed observation on cholesterol increase in tocilizumab-treated RA patients, the interest on impact of various biologic and non-biologic DMARDs increased significantly. tocilizumab, an IL-6 receptor inhibitor, increases circulating LDL levels [77], but no increased risk of major cardiovascular events was observed [78,79]. It is established that tocilizumab reduces the LDL hypercatabolic state and diminishes the expression of LDL receptor on hepatocytes via a proprotein convertase subtilisin/kexin type-9-mediated mechanism [80,81]. Quite recently, Greco et al. showed that treatment with tocilizumab improves the activity of scavenger receptor class B member 1 and ATP binding cassette-G1 (ABCG1) which directly leads to favorable modifications of lipoprotein composition and functions in contributing to the reduced cardiovascular risk in tocilizumab-treated patients [82]. Therefore, contrary to the general population, changes in LDL composition are not translated to increased cardiovascular risk in patients with RA.
