5.2.1. JAK Inhibitors and Lipid Profile

In the rabbit model, treatment with tofacitinib decreased systemic and synovial inflammation and increased circulating lipid levels have been observed. In this study, it failed to modify synovial macrophage density, however it reduced the lipid content within synovial macrophages. The study also confirmed the role IFN-γ in formation of foam cells, representing the key element for development of atherosclerosis. Inhibition of JAK/STAT pathway by tofacitinib contributed to lipid release via enhanced expression of cellular liver X receptor α and ATP-binding cassette transporter (ABCA1) synthesis [83]. Similarities in mechanisms of action in those two studies may suggest the role of IL-6 as a mediator of changes in lipid metabolism. The final effect may therefore be achieved by either IL-6 or JAK blockade (transmitting signals form IL-6-dependent receptor). Indeed, in an ex-vivo experiment, it was shown that tofacitinib decreased the expression of the IL-6 gene, having instead a variable effect on that of IL-8, TNF-α, and IL-10 genes [84]. Specifically, with ablation of JAK1 tofacitinib reduced signaling via IFN thus reducing TNF-α synthesis within macrophages. In a study with Chinese RA patients, the serum levels of TNF-α, IL-17, IL-6, and IFN-γ significantly decreased after the treatment with tofacitinib, parallel to an increase of IL-35, with subsequent T-reg lymphocyte response [85].

The role of IFN-γ in the modification of blood lipoproteins and promoting atherosclerosis development is postulated for many years. At the current level of knowledge, IFN signaling via JAK/STAT pathway regulates more than 2300 genes [86]. Signaling via IFN brings many pathophysiological consequences. Being a key element in immune response, IFN is also engaged in lipid metabolism and atherosclerosis development (Figure 3).

**Figure 3.** Interferons after ligating to type II receptors activate JAK/STAT pathway resulting in foam cells formation leading directly to atherosclerosis development. That same pathway that transmits signals from interferons contributes to reduced expression of the liver X receptor and decrease synthesis of ATP-binding cassette transporter leading to pro-atherosclerotic lipid composition.

Specifically, it can induce oxidative stress, promote foam cell accumulation, stimulate smooth muscle cell proliferation and migration into the arterial intima, enhance platelet-derived growth factor expression, and destabilize plaque [87]. This makes the IFN and STAT/JAK pathway the potential target to treat atherosclerosis. A few animal studies confirmed therapeutic potentials of IFN blockade resulting in inhibition of atherosclerotic plaque progression, stabilization of lipid- and macrophage-rich advanced plaques in ApoE knockout mice, and reduction of atherosclerosis in the graft vessels and the aorta in mice heart transplantation models [88–90]. Unfortunately, the inhibition of IFN blockade in atherosclerosis has not been tested in humans yet and studies on IFN inhibition in other indications (Crohn diseases and SLE) have been terminated prematurely due to lack of efficacy [87]. Another interesting study compared RA patients and healthy volunteers focusing on kinetics of cholesterol metabolism following a six-week tofacitinib treatment. In the study, the authors observed a reduction of the cholesterol ester fractional catabolic rate with subsequent increasing of HDL cholesterol and LDL cholesterol levels [91].

To summarize, blocking the JAK/STAT pathway may offer some potential to stop/reduce atherosclerosis development. The increment in lipid level did not translate to increased risk for atherosclerosis and is believed to be due to the transposition between several lipid compartments but not due to increased lipid synthesis. Moreover, as we already learned from the study with baricitinib, hypercholesterolemia, which occurred in less than 10% of the patients, was a dose-dependent event. Cholesterol level tends to increase during the first 12 weeks of the treatment, followed by stabilization of total cholesterol, LDL, and HDL serum levels when the treatment was continued [92].

#### 5.2.2. Thromboembolic Events

Data from many clinical trials on JAK inhibitors suggesting increased risk of thromboembolic events in patients treated with JAK inhibitors. This risk is especially high in patients who already have risk factors for venous thromboembolic event (VTE), including history of cardiovascular disease, increased body mass, hypercoagulable states, neoplasm, and history of prior VTE, as well as patients receiving estrogens, patients undergoing major surgery, or patients with movement disabilities. The pathophysiological background of unprovoked thromboembolism, referring to both deep venous thrombosis and pulmonary embolism, is largely unknown, but it is believed to be linked with comorbidities, lifestyle, and risk factors. Recently, however, data on gene expression profiles provided a new insight into the pathogenesis of VTE. Among many mechanisms potentially involved into the progression of the diseases, leukocyte transendothelial migration and JAK-STAT signaling pathway and their related genes were found to be related with recurrent VTE [87]. Moreover, the regulatory network of recurrent VTE displayed that most of differentially expressed genes including intercellular adhesion molecule 1 and protein-tyrosine kinase 2-beta were regulated by STAT3. This may bring many clinical consequences as down regulated JAK/STAT related genes are directly linked to recurrent VTE, so the open question is whether continuation of JAK inhibitor after the first episode of VTE brings the hazard of recurrent VTE [87]. Contrary to this finding, Lu et al. suggested the role of the JAK2-STAT3 pathway in the regulatory network of platelet activation. According to the results from the study, collagen-induced platelet activation was mediated through the activation of JAK2-JNK/PKC-STAT3 signaling. So, the inhibition of this pathway may exert anti-platelet activity [88]. The opposite conclusions come from the study of Ayer et al., who investigated the role of mutation of JAK2 in the development of thrombosis in chronic myeloproliferative diseases, finding no relationship between thromboembolic events and JAK2 mutation [89]. Recently, the role of tissue factor (TF) and its regulatory mechanism attracted attention of many researchers. TFs may be regulated in several ways including this provided by heparanase. Heparanase (heparanase-1) is a mammalian enzyme (endo-β-D-glucuronidase) that degrades side chains of heparan sulfate [90]. Heparanase gene expression is regulated via many pro-inflammatory pathways (cytokines, reactive oxygen species, early growth response 1 transcription factor, and estrogens) and play a role as in the pathogenesis of several inflammatory disorders, such as inflammatory lung injury, cancer development, and chronic colitis [93]. Data from the literature suggests also the role of heparanase in the development of inflammatory arthritis including RA [93]. In line with it, dramatic hyperactivity of heparanase and angiogenesis gene expression in synovium of RA patients were found [94]. Data form patients with thalassemia showed very high activity of heparanase which in turn may activate the coagulation system, leading to thrombotic events. This is partially mediated by a high erythropoietin level which activates the JAK2 pathway. Therefore, the modulation of the JAK2 signaling pathway may help to reduce risk of thrombotic events mediated by heparinase [95]. The other beneficial effect that JAK inhibitors may exert on the coagulation pathway, reducing the risk of thromboembolic events, is the reduction of fibrinogen synthesis in hepatocytes. In this special instance, the effect is indirect and is related to total reduction of inflammatory response via inhibition of IL-6 signaling [96].

The potential mechanisms that lead to an increased risk of thromboembolic event in patients with RA treated with JAK inhibitors remain the subject of controversy. Blocking the JAK/STAT pathway may bring both harmful and beneficial effects depending on the pathophysiological environment. Therefore, it may be suggested that not JAK inhibition alone, but comorbidities and risk factors presented in RA patients working together may in some predisposing patients increase the risk of thromboembolic complications.
