Bioprosthesis in aortic valve replacement: long-term inflammatory response and functionality

Background The evaluation of long-term inflammatory response and function in postoperative patients with aortic valve replacement (AVR) deserves special analysis because it is important to try to prevent reoperation and improve durability and functionality of the prostheses. It is our objective Methods In this study, we included a cohort of patients with aortic valve damage treated by AVR with mechanical prosthesis, bio prosthesis and we included a control group. Results We found that IL-4 and osteopontin levels were higher in patients with mechanical vs biological prostheses (p=0.01 and p=0.04, respectively), osteoprotegerin (OPG) levels were decreased (p=0.01), women had lower levels of ET-1 and IL-6, (p=0.02) (p=0.04), respectively. Patients older than 60 years had decreased levels of IL-1ß p<0.001) and a higher concentration of IL-4 p<0.05). IL-1ß, OPG and TNFα were higher in patients with less than 5 years of evolution vs more than 10 years (p=0.004, p=0.02 and p=0.03, respectively). Factors such as age, gender, prosthetic and elevated IL-1B and ET-1 levels are associated with valve dysfunction prosthetic. These results indicate that the inflammatory involvement present prior to valve replacement may be perpetuated by various factors in the long term. Conclusions The findings provide us with the opportunity to effectively treat patients with AVR in the postoperative period, which could prolong the functionality of the bio prostheses. Trial registration number NCT04557345.


INTRODUCTION
Aortic valve disease affects more than 26% of adult patients over 65 years of age 1 ; the main indication for aortic valve replacement (AVR) is aortic stenosis (AS), an active biological process with similarities atherosclerosis. 2 It begins with a lesion in the valvular endothelium that promotes the accumulation of lipoproteins and infiltration by macrophages and T lymphocytes. These cells secrete tumour growth factor (TGF-α), interleukin 1-β (IL-1β) 3 and other cytokines that generate the synthesis of matrix metalloproteinases and promote local remodelling. 4 In parallel, osteogenesis occurs when resident interstitial aortic valve cells (AVICs) are activated to fibroblasts by tumour necrosis factoralpha (TNF-α) and IL-1β, cells differentiate to fibrotic tissue and osteoblast-like cells. This process is promoted by the action of IL-6 and IL-4, 5 as well as by other promoter factors such as Osteopontin (OPN), 6

WHAT IS ALREADY KNOWN ON THIS TOPIC
⇒ Inflammatory mechanisms, among other processes, are strongly associated with a higher risk of dysfunction after prosthetic valve implantation. ⇒ The inflammatory process has been little studied and there is no specific therapy during its evolution. ⇒ Inflammation throughout the postvalve implantation and its association with prosthetic dysfunction remains unclear.
WHAT THIS STUDY ADDS ⇒ Factors such as age, gender, regardless of the type of prosthesis material, influence the risk of longterm prosthetic dysfunction. There is a significant incident inflammatory state whose type of cytokines are present deserve attention to evaluate mechanisms of action.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
⇒ Prevention or delay through anti-inflammatory and specific therapy in subjects undergoing valve implantation independent of age, gender and type of prosthesis could reduce the risk of subsequent dysfunction. ⇒ Clinical trials in this regard and further histological and biomarker studies may be necessary to reduce prosthetic valve dysfunction.
axis (OPG), the receptor activator of nuclear factor κB (RANK) and its ligand (RANKL). 7 These processes perpetuate valvular calcification, progressive reduction of the aortic valve area (AVA). The treatment for severe AS is AVR; however, the inflammatory state will persist in half of the patients in the short term. 8 Among them are prosthetic material (titanium), 9 the haemodynamic flow profile, the biological prosthetic valve tissue (porcine or equine) or mechanical factors. 10 Long-term surgical success can be improved with preoperative and postoperative therapeutic measures.

Valvular heart disease
This study's objective was to evaluate the function and long-term inflammatory response in postoperated patients with AVR using bioprostheses or mechanical prostheses.

METHODS
A retrospective study in a cohort of postoperated patients with AVR. between January 1995 and January 2020. We included patients older than 18 years of age who met diagnostic criteria for severe valve dysfunction, 11 and the Heart Team decided to perform AVR using a biological or mechanical prosthesis. 12 All patients with signed informed consent format. Were excluded Patients with other cardiac intervention in addition to AVR, with significant coronary artery disease, immunodeficiency and oncological or rheumatic disease We included for the evaluation a control group with 80 healthy subjects matched by age and gender.
We extracted from the clinical record and performed a two-dimensional transthoracic echocardiogram (TTE) before surgery, following the evaluation and recommendations for measuring the heart chambers 13 to rule out structural abnormalities and valvular dysfunction. Through brachial venipuncture, blood samples were obtained for the quantification of inflammatory mediators. Preoperative and postoperative data were collected.
Enrolment did not imply additional diagnostic procedures to sampling and no alternative or additional treatments were performed. Data management was carried out in order to make identification of an individual patient and the need for consent was required.

Study design
Outpatient follow-up for valve diseases was performed after AVR for 1-25 years after the intervention. Clinical questioning, physical examination and TTE were performed following the recommendations for the evaluation of valve prostheses. 14 We obtained Blood samples by brachial venipuncture to quantify biomarkers with a prior signed informed consent form.
Determination of cytokines Ten mL of peripheral blood were centrifuged at 2500 rpm for 10 min at 4°C. Then, 500 µL aliquots of serum were prepared and stored at −75°C until cytokine determination. Subsequently, endothelin 1 (ET-1), IL-1ß, IL-1, IL-6, OPN, OPG, RANK, RANK-L and TGF-α were determined by an ELISA in DuoSet sandwich (R&D Systems, Two-dimensional transthoracic echocardiographic study TTE was performed and evaluated by two expert echocardiographers following the recommendations for cardiac chamber quantification 13 and evaluation of valve prostheses. 14 A Phillips EPIC 7 (Philips, Andover Massachusetts) ultrasound was used with an S5-1 (1-5 MHz) transducer. The effective orifice area (EOA) of the PrAV (aortic valve prosthesis) was calculated employing the continuity equation. The peak velocity (Pvel) and mean Gradient (MG) of the PrVA were obtained in a five-chamber projection. The pulmonary artery (PSAP) was calculated by adding the right atrium pressure to the maximum Gradient of tricuspid regurgitation.

Measurement of cytokines in valve prostheses
Cytokine levels are shown in table 2. In the analysis by gender and age, women had lower levels of ET-1 than men (0.53 vs 0.67 pg/mL, p=0.02), and there was a difference in IL-6 (p=0.04). Patients older than 60 years had decreased levels of IL-1ß (3-15 pg/cm 3 , p<0.001) and higher concentration of IL-4 (30-15 pg/mL, p<0.05). Differences in cytokine levels in biological and mechanical valves and the control group are shown in table 3. When comparing by the time of evolution dividing the patients in under 5 years, between 5 and 9 years or patients with more than ten years of placement of the PrAV, and after having performed the AVR, there was no difference in the ET-1 level (p=0.81). IL-1ß, OPG and TNFα were higher in patients with less than 5 years of evolution versus those with more than ten years (p=0.004, p=0.02 and p=0.03, respectively) in the post hoc analysis test.

Evaluation of prosthetic functionality
Prosthetic valve dysfunction occurred in 18 patients (22.5%), without a difference between patients treated with biological vs mechanical valve prostheses (p=0.96). The main complications were anterior paravalvular leak with a frequency of (15%).
The proportion of prosthetic valve dysfunction at 12 years after AVR is 75% for patients with bioprostheses (95% CI=0.49 to 0.93) and 50% for mechanical prostheses (95% CI=0.19 to 0.88). The Wilcoxon test did not show statistical significance between the percentages of prosthetic dysfunction over time (p=0.47). (Figure 2) The restricted mean prosthetic dysfunction was 11.4 years for patients with biological prostheses and 19.4 years for mechanical prostheses. The Cox proportional hazards analysis in post-AVR patients is shown in table 4. Prosthetic valve dysfunction is shown in tables 5 and 6. Patients who were implanted with locally manufactured bioprostheses (INC valve) did not show differences in cytokine levels, echocardiographic parameters or prosthetic valve dysfunction than imported bioprostheses (tables 5 and 7). Figure 2 shows survival of functionality and we included a online supplemental graphical abstract.

DISCUSSION
Calcification of the native aortic valve is present even after AVR. The pathogenesis is multifactorial; factors such as mechanical forces lead to endothelial dysfunction, 2 altered flow dynamics, 17 production of inflammatory cytokines by the prosthetic material (titanium) 9 and xenoantigens such as Galα3Gal and the corresponding anti-Gal antibodies contributing to valve damage. The participation of cytokines before AVR and after the intervention has been studied to define whether this inflammatory damage requires timely therapy to prolong prosthetic durability. 8 Among the cytokines studied, IL-4 activates collagen synthesis, promotes fibrosis progression, and inhibits inflammatory cytokines production. 18 Its secretion occurs in response to microorganisms, prosthetic material, volumetric or pressure overload. 19 In this study, elevated levels of IL-4 were found in patients treated with a mechanical prosthetic implant; however, this increase was not associated with prosthetic dysfunction, rheumatic heart disease, gender or the time to progress after PrVA placement. The elevation of IL-4 could be associated with the inflammation that occurs postsurgery, promoting the alternative activation of macrophages into M2 cells, increasing repair macrophages (M2), and decreasing when interacting with IL-10 and TGF-β. These changes contribute to valve tissue repair, and our results confirm this judgement.
We further found an increase in OPN in patients that received mechanical and biological valve prostheses without statistically significant difference. However, this finding had only been demonstrated in dysfunctional biological prostheses. 20 One explanation for this finding is the evidence that in calcified porcine aortic valves, there is an increase in OPN, which activates osteogenic signalling. 21

Valvular heart disease
We found low levels of OPG in mechanical prostheses; in relation, this finding has been found that low levels of OPG lead to an osteoclastic transformation of the valve, 22 and on the other hand, there is a negative correlation between native AVA and OPG. 23 Increased ET-1 and endothelin receptor-A levels have been identified in patients with native AS, 24 and endothelin A and B receptors are located on the leaflets' tips and surface. 25 There is a transient increase following myocardial damage after AVR and a concomitant diastolic dysfunction 26 ; however, this does not persist, and it decreases in conjunction with brain natriuretic peptide after improvement of the ventricular function due to decreased LV afterload. 27 In our work, the ET-1 level was similar between mechanical or non-functional biological prostheses. However, in mechanical prostheses with prosthetic dysfunction and the first 5 years after AVR, ET-1 was found to increase; this finding is like to previous studies in dysfunctional biological valves. 20 IL-1β induces inflammation through the inhibition of factor-κβ and AVICs 28 ; therefore, its participation in the extracellular matrix remodelling will condition the proliferation of interstitial cells and the expression of MPPs, 3 and also a dysfunction has also been found in the anti-inflammatory mechanism of the interleukin receptor antagonist 1β. In this research, the levels of IL-1β were similar in patients receiving biological and mechanical prostheses with and without dysfunction. However, in dysfunctional prostheses to a long-term time (more than 10 years), there was a decrease in IL-1β, which suggests that its participation is broad and varies according to comorbidities, the prosthesis material gender, and time of evolution.
Before AVR, the inflammatory process will continue and persist; however, anti-inflammatory therapy should be proposed after implantation. The transformation process of AIVCs leads to postoperative valve dysfunction since they change to a myofibroblast phenotype that is activated in the presence of transforming growth factor beta1 (TGF-β1). 29 Recently proposed therapies such as l-arginine prevent osteogenic differentiation of AVICs and reduce matrix calcification regarding therapeutics. This effect is obtained through the modulation of proteins involved in the cellular redox system, the extracellular matrix's remodelling, and the inflammatory activation of AVICs. 30 Prostheses' advantages and disadvantages are well defined, including inflammation in the early and late postoperative periods. Studies that include punctual monitoring still require exploration through systematic randomised clinical trials to improve valve prostheses' functionality.

CONCLUSIONS
The inflammatory process present after AVR is chronic and multifactorial. OPN and ET-1 are increased in mechanical prostheses. There are no differences in durability and prosthetic dysfunction; however, the increase in IL-1β and ET-1 is associated with a greater risk of  prosthetic valve dysfunction regardless of prosthetic valve type. Factors such as age, gender, inflammatory status and type of prosthetic material influence long-term dysfunction.
Contributors HS-O conceptualisation, investigation, data curation, investigation project administration, methodology, methodology, formal analysis and writing (original draft and editing) JV-B data source, RM-V project administration of laboratory writing (original draft and editing); JIF-P: data source, project administration, writing (editing); KSM-Z, data collection; VJ-R: data source from laboratory, writing (editing); SAC-V: data source from image, writing (editing); AA-G: data collection and data source from image, writing (editing); GF-A: data source from laboratory, graphical and writing (editing); VG-L: data source from laboratory, translation and writing (editing); IP-T: data source and writing (editing); TESP : work of elaboration of valves and biomedical engineering investigation; MES data source from laboratory, writing (editing); conceptualisation, investigation supervision, guarantor, investigation project administration, methodology, methodology, formal analysis and writing (original draft and editing) Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.

Patient consent for publication Consent obtained directly from patient(s)
Ethics approval This study involves human participants and was approved by Ethics Committee Number 19-1139. Participants gave informed consent to participate in the study before taking part.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request. The data are available on reasonable request to the corresponding author.
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