Endothelial dysfunction is a crucial and early manifestation of vascular diseases. It is characterized by the impairment of the relaxations induced by nitric oxide (NO) and other vasodilator compounds (like prostacyclin or the endothelium hyperpolarizing factor), while the endothelial vasoconstrictor factors are increased (Félétou and Vanhoutte, 2006). A pro-oxidant and pro-inflammatory vascular environment is another characteristic of endothelial dysfunction (Félétou and Vanhoutte, 2006). Endothelial dysfunction is linked to the early development of DM in both humans and experimental animal models, and associated to reactive oxygen species (ROS) over-production (Durante et al., 1988; Calver et al., 1992; Johnstone et al., 1993; Angulo et al., 1998; Rodríguez-Mañas et al., 2003a,b).

There is also increasing evidence suggesting that the development of an inflammatory environment in the vasculature by pro-inflammatory cytokines is followed by an impairment of endothelial function. Thus, acute systemic inflammation in response to Salmonella typhi vaccine produces a temporary but profound dysfunction of human arterial endothelium in both resistance and conduit vessels, which is related to cytokine production (Hingorani et al., 2000; Kharbanda et al., 2002). Moreover, it is now well established, either in experimental models and humans, that pro-inflammatory cytokines impair vascular reactivity in different vascular beds, including resistance vessels (Vila and Salaices, 2005).

Although the IL-1 pathway is considered at present a critical player in the pathophysiology of both T1DM and T2DM (Donath and Shoelson, 2011; Herder et al., 2015), the evidence on the impact of IL-1β on vascular function is still limited. In isolated resistance microvessels from non-diabetic animals, IL-1β produces endothelial dysfunction after different exposure times to the cytokine (Wimalasundera et al., 2003; Jiménez-Altayó et al., 2006; Vallejo et al., 2014). Interestingly, the impairment of endothelial function may occur even after a rather short incubation with IL-1β (30 to 120 min), when the possible pro-inflammatory responses triggered by the cytokine are not fully developed, as indicated by the lack of involvement of inducible pro-inflammatory enzymes that require de novo synthesis (Vallejo et al., 2014). This early endothelial dysfunction evoked by IL-1β is rather due to the IL-1 receptor-mediated activation of NADPH oxidase, which enhances superoxide anion (O2•–) production (Vallejo et al., 2014). Interestingly, NADPH oxidase over-activation has been linked to excess ROS generation and the development of atherosclerosis in the context of diabetic vasculopathy (Olukman et al., 2010; Gray et al., 2013). The pharmacological blockade of IL-1 receptors by anakinra permits to attenuate both NADPH activation and endothelial dysfunction induced by IL-1β in microvessels from non-diabetic animals (Vallejo et al., 2014).

Interestingly, the intraperitoneal administration of anakinra can also partially recover the endothelial dysfunction observed in experimental diabetes after 15 days of diabetes induction (Vallejo et al., 2014). The animals receiving the drug exhibited a clear improvement of endothelium-dependent relaxations that was paralleled by the normalization of NADPH oxidase activity in the vascular wall (Vallejo et al., 2014). Intriguingly, the circulating levels of IL-1β were not found to be enhanced (Vallejo et al., 2014), as previously reported by others in experimental models (Yazar et al., 2011) or in diabetic patients (Mooradian et al., 1991; Pereira et al., 2014). The local over-expression of IL-1β in the vascular wall may be responsible for a paracrine inflammatory response, similarly to that described in vitreous samples from patients with proliferative diabetic retinopathy (Patel et al., 2008). In addition, other long-lasting pro-inflammatory mechanisms induced by IL-1β can be involved in the endothelial dysfunction associated to experimental diabetes, since improved endothelium-dependent relaxation was observed after the pharmacological blockade of cyclooxygenase or the inducible form of nitric oxide synthase (iNOS) (Vallejo et al., 2014).

Chronic vascular inflammation is at the basis of atherosclerosis, which in turn accounts for life-threatening complications of DM such as AMI or stroke. The pro-inflammatory cytokines IL-1β and IL-1α are widely expressed in human and experimental atherosclerotic lesions (Frostegård et al., 1999; Liu et al., 2014) and a large body of preclinical data reveals that IL-1β plays a major role in the progression and rupture of atherosclerotic plaques (Chi et al., 2004; Isoda et al., 2004; Merhi-Soussi et al., 2005; Chamberlain et al., 2006; Frostegård, 2013; Singla et al., 2016). Thus, a rationale is set for the use of pharmacological IL-1β blockers as tools to delay the onset and progression of atherosclerotic lesions.

One of the earliest steps of atherosclerosis is the recruitment of leukocytes by endothelial cells via the expression of adhesion molecules such as ICAM-1 and VCAM-1. The saturated fatty acid palmitate, which is linked to a higher risk of type 2 diabetes and cardiovascular disease, promotes the release of IL-1β by monocytes and their adhesion to human endothelial cells (Shikama et al., 2015). This effect can be blocked by anakinra, which prevents the induction of adhesion molecules elicited by IL-1β (Shikama et al., 2015).

An imbalanced trafficking and distribution of intracellular cholesterol has been associated with defective signaling and vascular cell dysfunction (Schreiber, 2002). In cultured vascular smooth muscle cells, serum amyloid A, which is expressed in atherosclerotic lesions, promotes cholesterol trafficking via the release of IL-1β (Pessolano et al., 2012). This effect can be inhibited by the recombinant form of IL-1Ra (Pessolano et al., 2012), and presumably, by IL-1β blocking drugs, although this point still needs to be addressed.

Moreover, the dysruption of the endoplasmic reticulum (ER) homeostasis by high glucose or other factors has been identified over the last years as an important mechanism that underlies many complications of type 2 such as endothelial dysfunction, vascular inflammation and atherosclerosis (Lenna et al., 2014). Chronic ER stress leads to the so-called unfolded protein response (UPR), which ultimately seeks to restore ER function but can also lead to cell death and apoptosis (Lenna et al., 2014). In a murine model of type 2 diabetes, the intraperitoneal injection of anakinra during 4 weeks diminished ER stress and macrophage infiltration, and improved ischemia-induced neovascularization and blood flow as compared with untreated animals (Amin et al., 2012). These effects were further accompanied by a normalization in the blood levels of cholesterol and adiponectin without affecting glycemia (Amin et al., 2012).

It is also worth noting that the pro-inflammatory signaling elicited by IL-1β in human vascular cells can be exacerbated by high glucose. In human endothelial cells, the expression of adhesion molecules such as ICAM-1 and VCAM-1 induced by IL-1β or TNF-α is augmented in the presence of extracellular high glucose (Azcutia et al., 2010). Functionally, the synergy between IL-1β and high glucose leads to an exaggerated leukocyte-endothelial adhesion in vitro, together with enhanced rolling flux and emigration of leukocytes in animal experimental models in vivo (Azcutia et al., 2010). Similarly, in human vascular smooth muscle cells IL-1β triggers the activation of pro-inflammatory pathways such as the ERK 1/2- NF-κB- inducible nitric oxide synthase (iNOS) axis (Lafuente et al., 2008). Such an effect of IL-1β is exaggerated proportionally to the concentration of extracellular glucose (Lafuente et al., 2008). As an explanation for this synergy, it has recently been shown that IL-1β permits the entry of extra glucose across the plasma membrane of human vascular smooth muscle cells (Peiró et al., 2016). Part of this excess intracellular glucose is then driven by the pentose phosphate pathway to produce NADPH that in turn fuels the pro-oxidant enzyme NADPH oxidase. The over-production of ROS seems ultimately to be responsible for the exaggerated NF-κB activation and iNOS induction triggered by IL-1β under high glucose conditions (Peiró et al., 2016). By using anakinra, not only the pro-inflammatory and pro-oxidant signaling elicited by IL-1β in vascular cells was blunted but also, and importantly, its exacerbation by extracellular high glucose (Peiró et al., 2016).

Despite the promising pre-clinical data available to date (Figure 1), the ability of anakinra or other IL-1β blocking drugs to prevent or retard diabetes-associated experimental atherosclerosis remains to be better established and requires additional research with in vivo models. The scarce data available on IL-1 blockade and atherosclerosis in patients suffering from T2DM will be presented later in this review.

Type 2 diabetes, together with obesity, has been shown to accelerate aging processes and, particularly, vascular aging (Minamino and Komuro, 2007; Barton, 2010). Indeed, besides presenting endothelial dysfunction and a pro-inflammatory status, animal models of obesity or diabetes exhibit core hallmarks of vascular aging and cardiovascular pathologies, such as arterial stiffness, calcification, and endothelial cell senescence (Brodsky et al., 2004; Sloboda et al., 2012). In humans, different reports have evidenced that patients suffering from type 2 diabetes display a higher propensity to calcified arteries, especially in the vasculature of lower extremities and in the coronary artery (Chen and Moe, 2003; Zhu et al., 2012), while they also exhibit enhanced arterial stiffness (Laugesen et al., 2013). The presence of senescent endothelial cells has been demonstrated in atherosclerotic lesions from aortae and coronary arteries of rat models of diabetes (Chen et al., 2002; Minamino et al., 2002). Vascular senescence is associated to a series of morphological and metabolic disturbances that result in dysfunctional cell homeostasis (Erusalimsky, 2009) The senescence-associated secretory phenotype is characterized by enhanced cytokine secretion that in turn drives local inflammation and vascular dysfunction (Erusalimsky, 2009). Overall, the occurrence of vascular premature aging in patients suffering from obesity and/or T2DM favors the development of vascular disease, markedly enhances cardiovascular risk and worsens the life expectancy of these patients. To date, there are few studies available on the impact of IL-1β on accelerated vascular aging. It is known, however, that it can promote both alkaline phosphatase expression and mineralization in cultured vascular smooth muscle cells, as two mechanisms favoring vascular calcification (Lencel et al., 2011). We have also recently observed that IL-1β can induce the accumulation of senescence-associated β-galactosidase in cultured human endothelial cells, an effect that can be dampened by anakinra (Peiró et al., unpublished results).

Heart failure (HF) is a complex clinical syndrome characterized by impaired cardiac function (left ventricular ejection fraction less than 40%), and enhanced inflammation which is associated with worsening outcomes in these patients. Although infection with microorganisms is not involved in the development of HF in most cases (sterile inflammation), inflammation has been implicated in the pathogenesis of HF. In this regard, IL-1β exerts crucial effects on most cell types involved in cardiac repair and injury, and beneficial and detrimental effects of IL-1β have been reported (Bujak and Frangogiannis, 2009). The pro-inflammatory and pro-fibrotic responses after myocardial injury serve to clear the wound and facilitate wound healing and scar formation, and an excessive inhibition of the inflammatory response can develop a defective scar (Frangogiannis et al., 2005). Also, in myocardial ischemia-reperfusion (I/R) injury, IL1β and IL1α pretreatment reduces ROS generation and a subsequent increase in glucose-6-phosphate dehydrogenase activity, which provides cytoprotective effects against oxidation (Hedayat et al., 2010). However, excessive upregulation or chronic stimulation of IL1β and IL1α have been linked to deleterious responses.

Several reports have demonstrated the benefit of blocking IL-1β in experimental models of HF. Early inhibition of IL-1β signaling is more likely to inhibit the inflammatory cascade, whereas late inhibition may predominantly abrogate the direct actions of IL-1β on fibroblasts (Bujak et al., 2008). In rodents, by using genetically engineered antibody to IL-1β, cardiac enlargement and dysfunction following AMI were reduced without affecting the infarct size (Abbate et al., 2010). By administration of a recombinant IL-1Ra in T1DM-induced diabetic cardiomyopathy, the IRAK2/CHOP-dependent apoptosis was attenuated, without affecting fasting blood glucose concentration (Liu et al., 2015). Interestingly, in T1DM-induced diabetic cardiomyopathy, administration of an anti-TNF-α monoclonal antibody lessen cardiac TNF-α and IL-1β expression in correlation with cardiac collagen-I and -III content, and improvement of left-ventricle function (Westermann et al., 2007).

Other strategies to control IL-1β activity in the context of cardiomyopathy address the kallikrein–kinin system and the NLRP3/caspase-1/TLR pathway. Transgenic activation of kallikrein-1 ameliorated intramyocardial inflammation through reduction of adhesion molecules, IL-1β and TNF-α, and leukocyte infiltration, as well as endothelium dysfunction and oxidative stress in T1DM-induced diabetic cardiomyopathy (Tschöpe et al., 2005). Moreover, NLRP3-deficient mice subjected to I/R exhibited a marked improvement of cardiac function and reduction of hypoxic damage (Sandanger et al., 2013). NLRP3 gene silencing ameliorated pyroptosis and ROS release under high glucose in cardiomyocytes. ROS inhibition decreased also NF-κB and mature IL-1β (Luo et al., 2014a). Finally, NLRP3 and high-mobility group box-1 (HMGB1) knockdowns reduced cardiac hypertrophy and fibrosis, and restored cardiac function (Fuentes-Antras et al., 2014). Ablation of TLR4 successfully reverted architectural aberrations and restored cardiac dysfunction in T1DM mice. Thus, the role of HMGB1 as TLR4 ligand and upstream inducer of NF-κB and NLRP3 may shape a key pathogenic axis in diabetic cardiomyopathy, suggesting their potential as novel anti-inflammatory approaches. In this context, other ligands for TLR (TLR2 and TLR4) such as biglycan could also play a key role in amplifying fibrotic responses after cardiac injury (Beetz et al., 2016).

In this regard, statins as rosuvastatin have also exhibited beneficial proprieties against diabetic cardiomyopathy through inhibition of NLRP3 inflammasome and mature IL-1β, via suppression of the MAPKs activation (Luo et al., 2014b). Similar effects for pravastatin were observed also in obese T2DM rats with AMI (Li et al., 2006). Moreover, administration of the PDE-5 inhibitor tadalafil reduced circulating IL-1β and TNF-α, and associated chemokines RANTES, MIP-1β and MCP-1, after I/R induction in T2DM mice. In parallel, tadalafil upregulated the anti-inflammatory cytokine IL-10 and improved fasting glucose, whereas decreased infarct size (Varma et al., 2012). In addition, a glucocorticoid (methylprednisolone) treatment reduced expression of TLR4/NF-κB signaling and IL-1β, IL-6, TNF-α, and ICAM-1 on T1DM-induced diabetic cardiomyopathy with I/R injury (Hu et al., 2011). More importantly, the antiapoptotic property of anakinra has been demonstrated in models of I/R injury and AMI. This action was due to a decreased expression of pro-apoptotic mediators Bax, Bak, and caspase-1 and -3, which promote a reduction of infarct size and favorable ventricular remodeling (Abbate et al., 2008). Also, in T1DM mice, released IL-1β from cardiac macrophages stimulated with TLR2 and NLRP3 agonists, induced a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, and subsequent cardiac arrhythmia. Interestingly, inhibition of IL-1β signaling by either anakinra or NLRP3 inhibitor (MCC-950) reduced these effects (Monnerat et al., 2016).

Although the results obtained with anakinra were encouraging, still there are characteristics that make the drug unsuitable for long-term treatment of T2DM, such as a short half-life (anakinra should be administered on a daily basis to maintain adequate suppression of IL-1β) or injection-site reactions, in addition to being an expensive option. It is also worth to note that an anti-IL-1 strategy that saves IL-1α activity could offer safety benefits. In fact, genome-wide association studies recently suggested that long-term dual IL-1α/β inhibition could increase cardiovascular risk mediated, at least in part, through an increase in proatherogenic lipid concentrations (The Interleukin 1 Genetics Consortium, 2015). Therefore, reducing specifically IL-1β activity with longer-lasting agents would provide a therapeutic improvement in terms of sustained inhibition of IL-1β. In this context, the monoclonal antibody canakinumab achieves IL-1β inhibition, while preserving IL-1α activity. Also, the significantly longer half-life of canakinumab (Chakraborty et al., 2012) makes the drug more suitable for assessing outcomes that may require longer treatment times, such as cardiovascular ones.

In fact, canakinumab is currently being tested in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), which is the largest trial in progress for any anti-cytokine drug (ClinicalTrials.gov identifier: NCT01327846). The objective is to test whether canakinumab will reduce cardiovascular events in high-risk patients with T2DM who have high CRP levels (hsCRP > 2 mg/L) despite receiving optimal statin therapy. The trial is examining 17,200 individuals using three doses of the antibody in a placebo-controlled, randomized design in 146 centers. Although the primary outcome is based on the hypothesis that blocking IL-1β activity in these patients may also reduce the incidence in myocardial infarction and stroke, secondary end points include prevention and improvement of diabetes. CANTOS is an event driven trial due to be completed in 2017, when approximately 1400 cases of myocardial infarction, stroke, or cardiovascular death will have accrued (Ridker et al., 2011).

A preliminary proof-of-concept study has been already executed within the CANTOS trial; thus, 556 well-controlled type 2 diabetic subjects (baseline HbA1c of 7.4%) with high cardiovascular risk have been randomly allocated to receive monthly subcutaneous canakinumab at doses of 5, 15, 50, or 150 mg or placebo and followed over 4 months. Together with an important dose-dependent reduction in inflammatory markers (CRP and IL-6), there was a modest non-significant improvement in glucose control parameters (HbA1c, glycemia, and insulinemia), while lipoprotein levels were unchanged (Ridker et al., 2012). In terms of cardiovascular outcomes, there is great expectation for the results of the trial. If positive, CANTOS would reinforce the inflammatory hypothesis of atherothrombosis and support a cytokine-based therapy for the secondary prevention of cardiovascular disease in T2DM.

Apart from the expected results from CANTOS, there are very few studies available on vascular outcomes in patients with DM and most of them show no clear cardiovascular improvement upon IL-1β blockade. A smaller trial enrolled 189 patients with atherosclerosis in the carotid artery and/or the aorta and either T2DM or impaired glucose tolerance (ClinicalTrials.gov identifier: NCT00995930). The subcutaneous administration of canakinumab (150 mg) monthly for 12 months did not significantly affect vascular structure in terms of carotid intima thickness, or vascular function in terms of aortic distensibility measured by pulsed wave velocity (Choudhury et al., 2016). Canakinumab showed an anti-inflammatory effect by reducing the circulating levels of IL-6 and hs-CRP, but had no effect on metabolic indicators, like fasting glucose, HbA1c, or insulin sensitivity (Choudhury et al., 2016). Although no cardiovascular benefit was found, the results were not considered conclusive due to some limitations, including the short duration of the study to assess structural changes. Also, the lack of stratification of the patients enrolled based on their initial inflammatory status did not permit to identify those patients that would potentially benefit more from IL-1β inhibition. Finally, the dose administrated was sufficient to lower inflammatory markers but perhaps was not high enough to generate a maximal effect on atherosclerotic lesions.

Another prospective non-controlled pilot study was performed with only six patients with proliferative diabetic retinopathy, secondary to T1DM or T2DM, and receiving subcutaneous canakinumab (150 mg) every 8 weeks (ClinicalTrials.gov identifier: NCT01589029). No regression of retinal neovascularization was reported after 24 weeks of follow-up, although a positive effect on macular edema was observed (Stahel et al., 2016). Again, the dose of canakinumab may have been insufficient, with higher doses of 300 mg every 4 weeks being currently approved for uses such as systemic juvenile idiopathic arthritis.

A possible limitation for these studies, in addition to sample size or dosage, can be that the patients enrolled already exhibited advanced stage cardiovascular disease. Thus, parallel studies performed in diabetic patients without evidence of advanced vascular lesions would help shedding light on the capacity of canakinumab or other IL-1β blocking drugs to prevent or retard the onset of atherosclerosis or other vascular alterations in the context of DM.

Canakinumab was safe and well-tolerated in the above-mentioned cardiovascular trials, with no significant differences in adverse effects between treated and control groups (Choudhury et al., 2016; Stahel et al., 2016). The upcoming results of CANTOS will help shedding more light on the tolerability of long-term treatments with canakinumab.

In the context of T2DM, other studies not specifically designed to assess cardiovascular outcomes, have already provided information on the tolerability of canakinumab. A recent study pooled the data from trials that used different doses of the drug (Howard et al., 2014). These trials enrolled a total of 1026 patients with different routes of administration, treatment regimes and follow-up time (ClinicalTrials.gov identifiers: NCT00900146 and NCT00605475; Ridker et al., 2012; Rissanen et al., 2012; Hensen et al., 2013; Noe et al., 2014). The global analysis of the three trials demonstrated that canakinumab was safe and well tolerated over a treatment period up to 1.4 years at the four pooled doses evaluated, which was in agreement with safety findings reported in the individual studies (Howard et al., 2014). No significant differences were evidenced in terms of adverse effects, discontinuations or deaths between treatment and placebo (Howard et al., 2014). Overall, canakinumab seems safe and well tolerated by patients with T2DM. However, additional trials with longer follow-up times are still needed to assess whether the drug will be suitable for longer-term treatment of a chronic conditions such as T2DM.