Antagonizing IL-17A Reduces Vascular Inflammation and Attenuates Oxidative Stress Formation but Does Not Significantly Improve Vascular Dysfunction Induced by One Week of Angiotensin II Treatment

TL;DR

Blocking IL-17A reduces vascular inflammation and oxidative stress but does not significantly improve vascular dysfunction caused by Ang II treatment in mice.

Contribution

The study investigates the early effects of IL-17A antagonism on Ang II-induced vascular dysfunction, revealing limited therapeutic benefit in this context.

Findings

IL-17A deficiency or anti-IL-17A treatment reduced oxidative stress and vascular inflammation after one week of Ang II.

These interventions did not significantly prevent Ang II-induced vascular dysfunction.

Anti-inflammatory therapies alone may not be sufficient to address vascular impairment in IL-17A-driven diseases.

Abstract

Introduction: The pro-inflammatory cytokine interleukin-17A (IL-17A) has a key role in the inflammatory cascade and promotes vascular inflammation and dysfunction. In addition, IL-17A is centrally involved in several autoimmune diseases. IL-17A deficiency has been linked to reduced vascular inflammation associated with attenuated arterial hypertension under long-term angiotensin II (Ang II) exposure for four weeks. This is of interest as IL-17A is one factor linking several autoimmune diseases with cardiovascular comorbidity. So far, little is known about the effects of IL-17A during the early stages of vascular dysfunction development—an interval possibly representing an optimal therapeutic window. Methods: Mice lacking the IL-17A receptor alpha (IL-17RAdel) and wild-type counterparts were treated with Ang II for one week (1 mg/kg bodyweight/week). We assessed systemic oxidative stress formation and vascular function, as well as inflammatory cells in the vessel wall. In parallel, C57BL/6J mice treated with Ang II received anti-IL-17A therapy, to evaluate the same parameters. Results: Both IL-17RA-deficient mice and anti-IL-17A-treated C57BL/6J mice exhibited an attenuated oxidative stress response and mitigated vascular inflammation following one week of Ang II treatment. These effects did not significantly prevent the onset of Ang II-induced vascular dysfunction at that timepoint. Conclusions: After one week of Ang II treatment, antagonizing IL-17RA or IL-17A only partially reduced/attenuated the Ang II-induced effects on the vasculature. In the context of IL-17A-driven autoimmune diseases with associated vascular pathology, our findings suggest that anti-inflammatory therapies alone may not be sufficient to attenuate vascular impairment. A combined approach including agents with direct protective vascular effects may be required for effective intervention for the associated vascular comorbidity.

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Full text

1. Introduction

Vascular dysfunction arises from the complex interplay of factors, including inflammation, hormonal regulation, endothelial–vascular interactions, and metabolic stress [1,2]. Additionally, crosstalk between the vasculature and other organs, such as the kidneys and skin, is essential for blood pressure regulation and endothelial homeostasis [3]. Importantly, immunological processes—encompassing both the innate and the adaptive immune systems—significantly contribute to the gradual development of vascular dysfunction. Myeloid cells (neutrophils, macrophages, and monocytes) providing phagocytic NADPH oxidase are a key source of reactive oxygen and nitrogen species (ROS/RNS). They contribute substantially to vascular dysfunction triggered by one-week Ang II treatment, a widely used mouse model of hypertension and vascular impairment [4]. Inflammatory processes driven and sustained by myeloid cells disrupt the balance between oxidative and anti-oxidative mechanisms in the vasculature [4]. This imbalance leads to vascular dysfunction and creates a vicious cycle of ongoing vascular damage [4,5].

T cell-derived IL-17A has a key role in inflammation by recruiting myeloid cells to the site of injury [6,7]. Therefore, it is not surprising that IL-17A participates in the pathogenesis of vascular dysfunction and inflammation. IL-17A treatment evoked an increase in the inhibitory endothelial Nitric Oxide Synthase (eNOS) Thr495 phosphorylation in endothelial cells and decreased NO-dependent relaxation responses in isolated mouse aortae [8]. Furthermore, IL-17A induced ROS/RNS formation in endothelial cells of the blood–brain barrier [9]. We could show that T cell-derived IL-17A elicits vascular dysfunction without a concomitant rise in blood pressure [10]. In the context of atherogenesis, IL-17A inhibition was associated with a reduction in the atherosclerotic lesion size in ApoE-/- mice [11]. We and others have shown that Ang II-induced vascular dysfunction is associated with increased IL-17A and RAR-related orphan receptor-γ (RORγt), as an essential transcription factor for Th17 differentiation in the aortic vessel wall, in analogy to the induced vascular dysfunction and inflammation [12]. In a mouse model of dermal IL-17A overexpression mimicking severe psoriatic disease, we could show that chronic IL-17A exposure leads to vascular dysfunction and vascular inflammation—and that a psoriasis-induced dermal barrier defect impacts various ways of vascular function and regulation [13]. Among these effects, we also found an increase in Ang II in the blood of these mice [13] in parallel to the elevated IL-17A driving potentially a vicious cycle contributing altogether to the cardiovascular comorbidity seen in psoriasis.

The Ang II model of murine hypertension is one of the most widely used experimental models for studying hypertension and associated vascular dysfunction [4]. It involves the continuous infusion of Ang II, typically via osmotic minipumps, to induce elevated blood pressure, vascular inflammation, oxidative stress, and vascular dysfunction [14,15]—key features of human hypertension. An influence of IL-17A in this model was established by Madhur et al., who showed that IL-17A deficiency reduced vascular inflammation and dysfunction in mice following long-term Ang II infusion over four weeks [16]. In a subsequent study, the same group found that monoclonal antibodies targeting IL-17A and its receptor (IL-17RA)—therapies already used for psoriasis and other autoimmune diseases—attenuated vascular inflammation and reduced end-organ damage under long-term Ang II exposure [17].

Building on data from long-term Ang II infusion studies, we sought to examine the role of IL-17A in the early phase of vascular dysfunction and inflammation. This was our goal as prevention and early treatment options need a better understanding of early changes in disease development. Therefore, we used the classical short-term Ang II model with only one week of Ang II treatment to assess the initial steps underlying vascular impairment. Specifically, we analyzed the impact of IL-17RA deletion and of anti-IL-17A treatment on the development of early Ang II induced vascular inflammation and dysfunction.

2. Materials and Methods

Mice and treatment: Tie2-IL-17A^ind/+^ mice were generated by crossing the Tie2-Cre mouseline [18] with the IL-17A^ind/ind^ mouse [19,20]. Cre-mediated recombination leads to the overexpression of IL-17A in endothelial cells (Tie2-IL-17A^ind/+^ [20]). Cre-negative IL-17A^ind/+^ mice, which do not show any phenotype alterations comparable to wild-type mice, served as control mice.

IL-17RA-knockout mice ubiquitously lack the expression of IL-17RA (IL-17RA^del/del^, in short form IL-17RA^del^ mice) [21]. As controls, C57BL/6J mice were used.

For the experimental hypertension model, either male 12–14-week-old C57BL/6J or IL-17RA^del/del^ mice were infused for 1 week with AngII (1 mg/kg bodyweight per day; Sigma-Aldrich, Co.; Saint Louis, MO, USA) via the implantation of Alzet^®^ osmotic pumps (model 1007D; Cupertino, CA, USA) or sham operated under isoflurane narcosis as described previously [4]. Mice were euthanized using an isoflurane inhalation overdose.

We used C57BL/6J male mice for all experiments, between 8–12 weeks of age, that were housed identically, except for the Tie2-IL-17A^ind/+^ mice and their control mice, which were younger (around 6 weeks). Mice were generally housed in the animal facility of the University Medical Center Mainz TARC (Translational Animal Research Center, Mainz, Germany) in accordance with the FELASA guidelines and approved by the TARC and the Animal Care and Use Committee (IACUC) from the Land of Rhineland-Palatine (RLP), Mainz, Germany (License numbers 23 177-07/G 10-1-019/approval date 8 June 2010 and G 15-1-051/approval date 22 March 2016). We regularly controlled the experimental mice concerning the weight (loss), behavior, pain, and general appearance and followed strict protocols and scoring systems in order to detect and reduce stress for the animals.

Anti-IL-17A treatment: To neutralize IL-17A in vivo in the Ang II hypertension model, C57BL/6J mice with Ang II osmotic pumps were treated either with 60 µg/g bodyweight of an anti-IL-17A antibody (BZN035) or isotype control antibody (ISO, ACE31966), both kindly provided by Novartis (Basel, Switzerland). Ang II osmotic pumps were implanted on day 0 (1 mg/kg bodyweight per day as described above). Anti-IL-17A or isotype treatment took place on days 0, 3 and 6. The final analysis of mice was accomplished on day 7.

We assessed and analyzed vascular function/dysfunction, ROS/RNS formation, and (vascular) inflammation in these mice.

Isometric tension studies: The thoracic parts of isolated aortas (4 mm) were liberated from fatty tissue and then cut into 4 mm segments. They were carefully rinsed to be completely free from blood inside the vessel. The endothelium-intact segments were put on force transducers (Kent Scientific Corporation, Torrington, CT, USA; Powerlab, ADInstruments, Spechbach, Germany) in organ chambers filled with Krebs–Henseleit solution 7 to perform concentration–relaxation curves of aortic tissue in response to increasing concentrations of acetylcholine (ACh) and glyceryl trinitrate (GTN) [4].

ROS/RNS measurement: The oxidative burst of the whole blood was quantified with L-012 (FUJIFILM Wako Chemicals U.S.A. Corp Richmond, VA, USA) enhanced chemiluminescence (ECL). Venous blood was taken from the heart, sodium citrate was added, and the blood was kept at room temperature. The L-012 ECL signal was counted with PDBu (10 μM) and without (basal) to detect the oxidative burst produced by blood leukocytes and was expressed as counts per minute [22,23]. If necessary, values were given as percentage values compared to control mice.

Blood pressure measurements: Systolic blood pressure was obtained in mice using a tail cuff non-invasive blood pressure system coupled to a PowerLab system (ML125 NIBP, ADInstruments) [20,24]. A minimum of three measurements were obtained from each mouse.

DHE immunofluorescent staining: For visualization of reactive oxygen species, we cut thoracic aortic cryosections of 4 μm thickness. They were rinsed and cleaned and then incubated in Krebs–Henseleit solution for 15 min at 37 °C and then embedded in Tissue Tec and frozen in liquid nitrogen. Then they were stained with the superoxide-sensitive dye dihydroethidium (DHE, 1 µM) over 30 min at 37 °C, based on our standard protocol [20]. Green autofluorescence from the aortic lamina and red ethidium fluorescence inside the ROS-producing cells were detected via fluorescence light microscopy.

Flow cytometric analysis: Single-cell suspensions from the aorta were prepared and incubated with Fc-block (BioXcell, Lebanon, NH, USA) to block non-specific antibody binding. Afterwards, cell suspensions were stained with monoclonal antibodies against the following surface antigens: CD45.2 (clone: 104, APC-eFluor780, eBioscience, San Diego, CA, USA), CD90.2 (clone: 53-2.1, APC-eFluor780, eBioscience or PerCP, Biolegend, San Diego, CA, USA), CD11b (clone: M1/70, PE-Cy7, eBioscience), F4/80 (clone: BM8, APC, Biolegend), Ly6G (clone: 1A8, PE, Biolegend), and Ly6C (clone: AL-21, BD Biosciences, Franklin Lakes, NJ, USA). The acquisition of stained samples was accomplished using the FACS™ Canto II (BD). Analysis was performed using Flow Jo Software (Software number 10.10, BD, Ashland, OR, USA). The gating strategy is described in Supplementary Figure S1.

Statistical analysis: Statistical analysis was performed with GraphPad Prism software (version 9; GraphPad Software, Inc., San Diego, CA, USA). Data were analyzed for a normal distribution (Kolmogorow–Smirnow test). When a normal distribution was given, we applied a Student’s t-test or one-way ANOVA test with a Tukey post-hoc test. If no normal distribution was given, the Mann–Whitney Test or Kruskal–Wallis test with Dunn’s multiple comparison or comparison of selected columns was used as appropriate and indicated in the figure legends. Aortic relaxation curves were compared using a two-way ANOVA multiple comparison with Bonferroni’s posttest.

p values of <0.001, <0.01, and <0.05 were considered statistically significant and marked by 3, 2, and 1 asterisks (*). Data are presented as mean ± SEM.

3. Results

Initially, we analyzed the vascular function in mice with endothelial IL-17A overexpression provoking local and direct IL-17A exposure of the vasculature to the cytokine of interest: (Tie2-IL-17A^ind/+^ mice). These mice showed a significantly reduced vascular function (Figure 1A) combined with increased systemic and vascular oxidative stress formation (Figure 1B,C) underscoring the harmful role of the cytokine IL-17A in vascular dysfunction development. Of note—and exactly as in psoriatic mouse models based on IL-17A overexpression in the skin—only the Acetylcholine (ACh)-induced (endothelium-dependent) vascular relaxation of the aortas resulted in significant impairment compared to control aortas (Figure 1A) [20]. The nitric oxide (NO)-induced (smooth muscle cell-dependent) [20] aortic relaxation remained unchanged.

To take the opposite approach, we subsequently analyzed the vascular function in mice lacking IL-17RA (IL-17ARAdel mice), the primary receptor for IL-17A signaling, to determine whether limiting IL-17A signaling could improve the vascular function. Although the ACh dependent vascular relaxation curve in IL-17RA^del^ showed a shift to the left compared to C57BL/6J control mice at baseline, there was no significant difference between the maximal relaxation of these curves (Figure 2A). Short-term Ang II treatment over one week (1 mg Ang II per kg bodyweight per day applied via osmotic mini pumps) evoked in both the IL-17RA^del^ mice, as in the wild-type mice, a significant right shift of the aortic relaxation curve as a sign of vascular dysfunction compared to littermates without Ang II infusion (Figure 2A). Also, here (similar to the baseline results), vascular function seemed to be slightly improved in Ang II treated mice lacking IL-17RA compared to Ang II-treated control mice—but this did not lead to a significant difference in maximal relaxation. However, ROS/RNS formation in blood was not significantly increased in response to Ang II in mice lacking IL-17RA as it is classically seen in control mice in response to Ang II (Figure 2B). As previously described [4], one week of Ang II treatment evoked the infiltration of myeloid cells (CD11b^+^, Ly6C^+^Ly6G^+^, Ly6C^+^Ly6G^−^ cells) into the aortic vessel wall of wild-type mice (Figure 2C, our current gating strategy is visualized in Supplementary Figure S1). This effect was significantly attenuated in mice lacking IL-17RA: in Ang II-treated IL-17RA-deficient mice, we observed no significant increase in CD11b^+^ myeloid cells, Ly6C^+^Ly6G^+^ neutrophil granulocytes, or Ly6C^+^Ly6G^−^ monocytes (Figure 2C). Measuring the blood pressure in both the IL-17RA^del^ mice and in the wild-type mice with and without one week of Ang II treatment, we found, in both strains, an increase in blood pressure under Ang II—but this increase was only significant in the control mice and not in the IL-17RA^del^ mice (Figure 2D). Therefore, the lack of IL-17RA did not protect from the development of vascular dysfunction induced by one week of Ang II treatment, but significantly reduced the myeloid cells infiltrating the aortic vessel wall and attenuated the increase in systemic ROS/RNS formation and the increase in blood pressure induced by one week of Ang II.

To neutralize IL-17A in vivo, C57BL/6J mice with Ang II osmotic pumps were treated either with 60 µg/g bodyweight of an anti-IL-17A antibody (BZN035) or isotype control antibody (ACE31966) and compared to C57BL/6J mice without Ang II treatment or with Ang II treatment alone. Anti-IL-17A treatment did not improve Ang II-induced vascular dysfunction (Figure 3A), similar to the findings in Ang II-treated IL-17RA-deficient mice. Nevertheless, anti-IL-17A treatment significantly attenuated Ang II-induced ROS/RNS formation in the blood (Figure 3B). In parallel, the numbers of CD11b^+^ myeloid cells, as well as Ly6C^+^Ly6G^+^ neutrophils and Ly6C^+^Ly6G^−^ monocytes, which were significantly increased in the aortic vessel wall after one week of Ang II treatment, were reduced, based on a trend, under applied anti-IL-17A antibody treatment (Figure 3C).

In summary, endothelial IL-17A overexpression induced significant vascular dysfunction. The absence of IL-17RA markedly reduced vascular inflammation and attenuated oxidative stress formation and the blood pressure increase induced by one week of Ang II treatment. Anti-IL17A treatment significantly reduced systemic ROS formation and attenuated vascular inflammation based on the trend. Neither the absence of IL-17RA nor anti-IL17A treatment could significantly improve vascular dysfunction following one week of Ang II treatment.

4. Discussion

Our results underscore the direct detrimental effect of the cytokine IL-17A on vascular function. On the one hand, endothelial IL-17A overexpression led to significant vascular dysfunction. While the absence of IL-17RA did not significantly improve vascular dysfunction following one week of Ang II treatment, it markedly reduced vascular inflammation and attenuated systemic oxidative stress formation and the Ang II-induced blood pressure increase. Consistently, anti-IL-17A treatment did not improve vascular dysfunction induced by one week of Ang II treatment either but significantly attenuated oxidative stress formation and attenuated vascular inflammation. Therefore, we provide evidence that both anti-IL-17A therapy, as well as the deletion of IL-17RA, can impact in a protective effect on vascular inflammation and oxidative stress formation already in the early phase of Ang II-induced vascular dysfunction development.

Saleh and Madhur et al. have shown previously that anti-IL-17A treatment and IL-17A KO, respectively, improved vascular dysfunction and inflammation induced by longterm Ang II treatment over four weeks [16,17]. Moreover, chronic end-organ damage, including renal inflammation and injury induced by long-term Ang II treatment, was reduced when IL-17A signaling was disrupted [17]. The authors discussed the possibility that biologicals targeting IL-17A could be beneficial in autoimmune diseases associated with cardiovascular disease [17]. Although both the work of Saleh et al. and Madhur et al. [16,17] and our experimental approach used Ang II as an agent to induce vascular inflammation and dysfunction, the two approaches differ substantially in the duration of application: the Ang II model over four weeks is known to be a very robust inducer of sustained hypertension, inflammation, and vascular dysfunction with an already chronic component reflected by marked end organ damage [25,26]. It is one of the most often used murine models for preclinical hypertension research [27]. In contrast, the short-term administration in our experiment mimics a different disease state: it is known that the blood pressure effects of Ang II can be already found in minutes to hours after infusion [28,29]—cardiac hypertrophy or kidney injury as sites of hypertensive end organ damage need longer exposure—usually two to four weeks—of the organism to Ang II [27]. We used the one-week Ang II model to investigate the very early effects mimicking an early phase of vascular dysfunction and vascular inflammation—paving the way for atherosclerosis and cardiovascular disease [30]. Interestingly, our data indicate that IL-17RA KO attenuated early vascular inflammation induced by just one week of Ang II infusion without significantly improving vascular dysfunction. This might also explain why Madhur et al. could show that IL-17A KO significantly attenuated vascular dysfunction evoked by four weeks of Ang II treatment [16]. Therefore, over the course of time of Ang II treatment, the contribution of IL-17A to the Ang II-induced vascular dysfunction seems to strengthen and to become more relevant. This could be due to the fact that IL-17A itself evolves over time: in the beginning of the inflammatory stimulus, it is involved in the recruitment of myeloid cells and especially neutrophils to the site of inflammation to fuel the inflammatory cascade and in the switch from the innate to the adaptive immune response [6,31]. Over time, it contributes to keeping up the inflammatory response in the direct interaction with several other cytokines, such as IL-6, and in the interaction with the surrounding tissue and the vasculature/the endothelial cells [6,31]. IL-17A is relevant in protecting the host against pathogens and in maintaining physiological homeostasis—and, on the other hand, it drives autoimmune diseases [32]. Here, a certain balance is indispensable. The cytokine milieu, as well as the local microenvironment, such as a balance between immune cell types, at the site of infection or inflammation also influence the balance between pathologic and protective IL-17 responses [32]. Altogether, the local and systemic balance, time point, and microenvironment impact the role of IL-17A in general. With regard to the vascular system, this cytokine has to be kept in mind when treating patients with IL-17A-driven autoimmune diseases and cardiovascular comorbidity. And the best option is to start early when treating the autoimmune disease—and to treat the vascular comorbidity with protective agents, such as ACE inhibitors, also from a very early timepoint on.

Our results support the conclusion that IL-17A is already involved in vascular inflammation induced by one week of Ang II treatment, as evidenced by the significant reduction in Ang II-induced myeloid cell infiltration into the vessel wall in IL-17RA-deficient mice. However, vascular relaxation remained impaired following one week of Ang II treatment, indicating that this effect occurred via an IL-17A-independent mechanism—at least in this early phase of Ang II-induced vascular impairment. Numerous studies have documented the direct detrimental effects of Ang II on endothelial homeostasis, most of them using cultured endothelial cells [33,34,35]. Evidence suggests, for example, that Ang II-induced endothelial dysfunction involves the inducible nitric oxide synthase and cyclooxygenase-dependent production of vasoconstrictive molecules from the endothelium, independent of immune cell infiltration or the development of hypertension [36]. Of note, the role of IL-17A in murine atherosclerosis is controversial [37]: IL-17A deficiency did not affect the aortic plaque burden after a high-fat diet, although a pro-inflammatory effect on atherogenesis was described [38]. More than that, IL-17A could also contribute to limiting atherosclerotic lesion development and was associated with plaque stability in humans, as shown by Ziad Mallat and colleagues [39]—presenting the double-edged sword of this cytokine. Possibly, the balance of the inflammatory system in the vasculature in the process of time is a relevant factor paving the way for the development of vascular disease.

Certainly, our current study is only a mouse-experimental approach, and it addresses only one model of vascular dysfunction (one week of Ang II treatment in mice reflecting a rather short time model of Ang II treatment). As described above, our results can be compared to the already published results from Madhur et al. who analyzed IL-17A KO mice under four weeks of Ang II infusion and could show a preserved vascular function and reduced oxidative stress formation, as well as a significant blood pressure reduction, in the IL-17A KO mice under Ang II treatment compared to wild-type mice under Ang II treatment after an initial similar blood pressure increase response [16]. Further analysis will be necessary to gain a deeper understanding and especially mechanistic insights of the role of IL-17A in vascular dysfunction—also with regard to the time frame and potential fluctuations in IL-17A levels. This is relevant for patients with IL-17A-mediated autoimmune diseases and associated cardiovascular comorbidity.

In the context of IL-17A-mediated autoimmune diseases [40] associated with life-threatening cardiovascular comorbidities [41,42], it is particularly important not only to address the visible and predominant symptoms of the autoimmune disease but also to target the associated, often silent, cardiovascular complications [43]. Although Ang II is traditionally used as a murine model for arterial hypertension and vascular inflammation, we aim to interpret our findings in the broader context of IL-17A-associated autoimmune diseases and their cardiovascular complications, given the longstanding discussion of IL-17A’s role in hypertension and vascular dysfunction. Our data underscore the importance of assessing the treatment efficacy during the early stages of vascular dysfunction, as we observed that IL-17A blockade effectively inhibits vascular inflammation even in the initial phase. In patients with autoimmune diseases, symptom development and disease progression often follow a relapsing–remitting, wave-like pattern [44]. It is tempting to speculate that the inflammatory mechanisms driving vascular inflammation might also be wave-like—and not steadily progressive. If Ang II induced vascular inflammation can be attenuated by antagonizing IL-17A signaling during the early phase of vascular dysfunction, this may enhance the vasculature’s responsiveness to conventional cardiovascular medications. This approach could therefore be particularly relevant for patients with autoimmune diseases who experience associated vascular comorbidities.

There has been a long ongoing discussion of if anti-inflammatory treatment is a relevant therapeutic option in patients with clinically relevant cardiovascular disease and which cytokine is the main driver of vascular dysfunction and therefore could be a relevant target point for treatment options [45,46]. IL-17A has been specifically proposed as a druggable target not only in the context of vascular dysfunction but specifically for arterial hypertension [47,48]; however, this remains a theoretical concept without established relevance or applications in clinical practice. Beyond the still insufficient understanding of the pathophysiology of arterial hypertension, it needs to be kept in mind that various cytokines are important in the inflammatory orchestra impacting on the vasculature and therefore are potential target points. In consequence, these therapeutic options are not easy to choose and provide many pitfalls. Therefore, they are—at least up to now—still far away from the clinical routine. Anti-inflammatory drugs such as canakinumab and colchicine can both reduce the incidence of major cardiovascular events in ischemic heart disease patients, but only colchicine had acceptable safety for use in secondary cardiovascular prevention [45]. For primary prevention and early therapy in patients with (IL-17A driven) autoimmune diseases and cardiovascular comorbidities, the optimal timing and choice of anti-inflammatory therapy remain to be determined. Most likely, the most favorable therapeutic option is the combination of an immune-modulating drug for the treatment of the underlying autoimmune disease reducing also vascular inflammation with classical cardiovascular therapeutic agents with a protective effect on the vasculature.

5. Limitation

Our study is a murine approach and not a “from-bench-to-bedside” analysis—and this reflects the main. We have focused on the analysis of one week of Ang II treatment in mice and have not yet performed further analysis on IL-17A in Ang II-induced vascular dysfunction and inflammation over a shorter or longer period of time of Ang II treatment or exact time point analysis. However, our results can be correlated with the publication of Madhur et al. [16], in which the impact of IL-17A KO in mice under four weeks of Ang II treatment was analyzed: here, vascular function was significantly improved, and blood pressure and vascular inflammation were reduced [16]—and this gives a hint for the growing relevance of IL-17A with increasing time of Ang II treatment. The role of IL-17A is controversial in atherosclerosis—it seems to have a plaque-stabilizing effect in the early phase but contributes to vascular inflammation in atherosclerosis [38,39]. This altogether underlines that further research is relevant to gain a better understanding of the contribution of IL-17A and associated cytokines to vascular disease, especially with regard to the time frame and the surrounding circumstances. Another limitation of our study is the missing direct mechanism and the analysis at which time point and via which (direct and indirect) pathways IL-17A and anti-IL17A impact the vasculature. An analysis of vascular function in humans with autoimmune diseases with and without anti-IL-17A treatment could potentially contribute to reach further knowledge on the exact impact of the cytokine IL-17A (in the early phase of) on vascular dysfunction development in humans. And this altogether can pave the way for a more individualized patient care.

6. Conclusions and Clinical Outlook

Our results suggest that IL-17A is already of relevance for the early phase of Ang II-induced vascular inflammation—although less pronounced compared to longer Ang II treatment. Deletion of IL-17RA significantly reduced vascular inflammation induced within one week of Ang II treatment and attenuated ROS formation. Anti-IL17A treatment attenuated vascular inflammation based on a trend and significantly reduced ROS formation. These findings could give hint to the potential dual benefit of anti-IL-17A/anti-IL-17RA-based therapies in autoimmune diseases, which are themselves often associated with significant cardiovascular comorbidity: besides their approval as essential therapy for various IL-17A-driven autoimmune diseases, these therapies seem to have a protective impact on the inflammatory situation already in the early phase of vasculature dysfunction development. Nonetheless, we must be aware that the antagonization of IL-17A after one week of Ang II treatment attenuated vascular inflammation but did not mitigate vascular dysfunction. Therefore, the best therapy for autoimmune diseases associated with cardiovascular disease would be a combination of anti-IL-17A with cardioprotective therapy and to treat the cardiovascular component from an early timepoint on.