Plasma endothelin-1 levels in patients with rotator cuff tear: a case-control study

Introduction

To date, we know that the etiology of rotator cuff tears (RCT) is multifactorial. Various studies propose a role for genetics in the pathological process, as well as the comorbidities that the patient presents (1-3). From an epidemiological perspective, it has been observed that the incidence of RCTs increases with advancing age (4), and it is more common in patients with comorbidities such as hypertension, diabetes mellitus, dyslipidemia (5), and obesity (6). Furthermore, some studies conducted on patients with RCTs suggest that lifestyle may play an important role in the pathogenesis. The incidence of RCTs appears to be higher in smokers (7,8) and in those who consume large quantities of alcohol (9), especially if these behaviors persist over time.

The combination of these intrinsic and extrinsic factors appears to act at a vascular level, resulting in endothelial dysfunction, including in the vascularization of the rotator cuff (10). Indeed, altered vascularization at the osteo-tendinous interface seems to play a significant role in this tendon degeneration.

This has been anatomically and histologically demonstrated over time; firstly, Lindblom [1939] (11) and then Moseley and Goldie [1963] (12) identified a region of hypovascularity near the insertion of the supraspinatus tendon, which was termed the “critical zone”.

Brooks et al. identified this area of hypovascularity within the distal 15 mm of the supraspinatus and infraspinatus (13). Rothman and Parke [1965] described this critical zone as the area where most supraspinatus tendon tears tend to develop (14).

Rathbun and Macnab conducted a study on cadavers to better visualize the vascular bed of the rotator cuff, which particularly emphasized the avascular critical zone of the supraspinatus, which was consistently present even in cadavers of young individuals around the age of 20 years (15).

On the molecular side, it has been studied that abnormal levels of endothelin-1 (ET-1) are associated with pathological conditions where endothelial dysfunction is observed, such as hypertension (pulmonary and systemic), atherosclerosis, heart failure, renal insufficiency, cerebrovascular diseases, and more (16-18).

ET-1 is a potent endogenous vasoconstrictor. It is a 21-amino acid polypeptide primarily synthesized by vascular endothelial cells from the EDN1 gene. This synthesis increases when these cells are exposed to substances such as growth factors and inflammatory cytokines, insulin, but also in response to vasoactive substances like norepinephrine, angiotensin II, and oxidized low-density lipoproteins (LDL). Conversely, its expression is reduced by vasodilatory elements like nitric oxide (NO), prostacyclins, and atrial natriuretic peptide (16-18).

ET-1 acts by binding to two receptors, ET-A and ET-B, which have opposite functions. ET-A promotes vasoconstriction, inflammation, and cell proliferation, while ET-B acts as an antagonist to ET-A, stimulates the release of NO and prostacyclin (resulting in vasodilation), prevents apoptosis, inhibits the expression of endothelin converting enzyme (ECE) in endothelial cells (an enzyme necessary for the maturation of ET-1), mediates pulmonary clearance of ET-1, and the reuptake of ET-1 by endothelial cells (16-18).

ET-1 acts in an autocrine and paracrine manner, performing multiple functions. It has inotropic, proinflammatory, and mitogenic roles, is involved in regulating the hydroelectrolytic balance (stimulating the renin-angiotensin-aldosterone system) and the stimulation of the sympathetic nervous system. Its primary role, however, is as a regulator of vascular tone, which is essential in both physiological and pathological conditions.

Our aim was to assess for the first time whether patients who have a RCT also exhibit altered levels of ET-1, as this peptide is a potent vasoconstrictor. We conducted a case-control study in which we compared plasma levels of ET-1 in patients with RCT to a population of healthy controls to assess potential alterations in plasma ET-1 levels. The article is presented in accordance with the STROBE reporting checklist (available at https://aoj.amegroups.com/article/view/10.21037/aoj-24-24/rc).

Methods

A case-control study was conducted. Cases consisted of 60 patients (28 women, 32 men) who underwent arthroscopic repair of RCT. The cases’ mean age was 59.65 [standard deviation (SD), 7.19] years, range 43–75 years. The tear had been diagnosed through a detailed medical history, physical examination and both X-rays and magnetic resonance imaging (MRI). Confirmation of tear severity was obtained during arthroscopic surgery.

The exclusion criteria are as follows: acutely traumatic cuff tears; a previous operation on the shoulder; inflammatory joint disease; osteoarthritis in the operated shoulder or in the contralateral (non-operated) shoulder; use of anti-inflammatory drugs in the two months prior to the surgery.

For the intraoperative classification of the lesion, the Southern California Orthopaedic Institute classification (19) of complete RCT was used, considering type I lesions as small, type II and III as large ones, and type IV as massive ones.

Controls consisted of 43 individuals (26 women, 17 men) voluntarily recruited from healthcare workers of the same hospital where arthroscopic repair was performed. Controls mean age was 53.18 (SD, 5.92) years, range 41–67 years.

The individuals underwent a comprehensive physical examination to assess the integrity of the rotator cuff, conducted by the more experienced authors; both the anterior and posterosuperior cuff tendon integrity were investigated. The integrity of the rotator cuff was also assessed using MRI. A positive result in any of these physical tests or MRI led to the exclusion of some of these controls. Additional exclusion criteria, in addition to those already mentioned for the cases, included the following: smoking habit, alcohol consumption habit, the presence of various comorbidities (hypertension, diabetes, heart failure, etc.).

Each participant was informed about the study’s methods and purposes and signed an informed consent through which they agreed to provide a peripheral venous blood sample for later analysis. For all participants, peripheral venous blood samples were collected.

Samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA), centrifuged for 15 minutes at 1,000 ×g at 2–8 ℃ within 30 minutes of collection, and then stored at a temperature ranging from −20 to −80 ℃ before analysis.

The analysis of the samples was performed using a specific sandwich enzyme-linked immunosorbent assay (ELISA) kit for human ET-1 (Human Endothelin 1 ELISA Kit, Catalogue No. A3318, antibodies.com).

The kit used contains wells in which specific antibodies for human ET-1 (primary antibodies) are present. In each well, a plasma sample taken from the cases or controls was added. ET-1 in these plasma samples binds to the antibodies in the wells, forming an antigen-antibody immunocomplex. After an incubation period necessary for the formation of these immunocomplexes, a washing step was performed to remove anything that was not bound by the primary antibodies. Subsequently, biotin-conjugated secondary antibodies were added to each well, which bind to the immunocomplex previously formed.

At this point, avidin conjugated with horseradish peroxidase (HRP) was added to each well since avidin binds biotin with high specificity and affinity. This is essential for the colorimetric reaction to occur. To initiate the colorimetric reaction, 3,3,5,5’-tetramethylbenzidine (TMB), which is an HRP substrate, was added to the wells. The enzyme-substrate reaction (between HRP as the enzyme and TMB as the substrate) is initiated by adding a solution of sulfuric acid.

The greater the quantity of ET-1 present in the plasma sample, the greater the magnitude of the colorimetric reaction.

The colorimetric reaction that follows is then measured using spectrophotometry at a wavelength of 450±10 nm. The concentration of ET-1 is determined by comparing the colorimetric reaction of the samples with a standard curve. This method allows for the quantification of ET-1 levels in plasma samples.

This kit allows for the detection of ET-1 concentrations ranging from 1.56–100 pg/mL, although it is possible to detect higher concentrations by diluting the sample. In this case, the result should be multiplied by the dilution factor. The kit has a sensitivity of 0.67 pg/mL. Additionally, using specific monoclonal antibodies, the specificity is high, and there have been no demonstrated cross-reactions or interferences between ET-1 and its analogs.

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of Sapienza University of Rome (No. 32/2021) and written informed consent was obtained from all patients.

Statistical analysis

To assess the normal data distribution the Shapiro-Wilk test was used. To evaluate the presence of statistically significant differences or not in terms of mean plasma ET-1 values among the examined groups, Mann-Whitney U test and analysis of variance (ANOVA) were used as needed. Calculated P values were 2-sided, a P value of less than 0.05 was considered significant, and the range of confidence interval (CI) was 95%, when appropriate. At least 19 patients for each group would be required, assuming a 2-tailed α value =0.05 (sensitivity =95%) and a β value =0.80 (with a study power of 80%). Statistical analysis was performed using G*Power 3.1.9.4 software (Heinrich-Heine-Universität, Düsseldorf, Germany).

Results

A total of 54 cases were recruited: 23 women, 31 men, mean age 59.69 (SD, 7.43) years, range 43–75 years. Among these, 28 had no comorbidities, with 7 of them being smokers and 21 being non-smokers. The remaining 26 cases reported comorbidities, including hypertension, dyslipidemia, asthma, or thyroid disorders (hypothyroidism). As for the control group, 25 subjects were recruited: 13 women, 12 men, mean age 53.28 (SD, 7.16) years, range 41–67 years. The 54 RCTs in the cases were classified as 24 small (44.44%), 8 large (14.81%), and 22 massive (40.74%). The characteristics of the studied group are reported in Table 1.

The mean plasma values of ET-1 in cases and controls were 81.85 (SD, 40.39) pg/mL and 83.28 (SD, 30.29) pg/mL, respectively (P=0.63, Cohen’s d=0.04) (Figure 1).

Figure 1 Mean plasma values of ET-1 in cases (blue) and controls (orange). ET-1, endothelin-1.

According to tear severity, the mean values of ET-1 were 88.39 (SD, 40.00) pg/mL, 72.07 (SD, 51.78) pg/mL and 78.27 (SD, 37.05) pg/mL in small, large and massive tears, respectively. No differences were found between the groups (P=0.54) (Figure 2).

Figure 2 Mean plasma values of ET-1 in cases with small tears (blue), large tears (orange) and massive tears (grey). ET-1, endothelin-1.

Further comparisons were made within the case group only.

Considering the comorbidities, no significant differences were found between patients with (mean ET-1 value ± SD =79.17±39.79 pg/mL) and without (mean ET-1 value ± SD =84.34±41.51 pg/mL) medical conditions (P=0.59, Cohen’s d=0.13). No differences were found comparing smoker patients (mean ET-1 value ± SD =84.09±65.78 pg/mL) with non-smokers (mean ET-1 value ± SD =84.42±32.07 pg/mL) (P=0.49, Cohen’s d=0.006). Comparing the controls (25 controls, mean ET-1 value ± SD =83.28±30.29 pg/mL) with cases presenting only a RCT, meaning non-smokers without comorbidities (21 cases, mean ET-1 value ± SD =84.42±32.07 pg/mL), a non-statistically significant difference was found with P=0.84, Cohen’s d=0.036. Comparisons between groups were summarized in Tables 2-4.

Discussion

It is widely documented that plasma levels of ET-1 are found to be higher in patients with various pathological conditions characterized by vascular endothelial dysfunction, such as arterial and pulmonary hypertension, heart failure, stroke, renal failure, and diabetes mellitus. However, it is not clear whether the elevation of ET-1 plasma levels, compared to healthy controls, is a cause or a consequence of the disease (16-18).

The etiology of RCT is multifactorial. Over the years, an increasingly important role has been attributed to the theory that underlying the tear is endothelial dysfunction, caused by intrinsic and extrinsic factors. The incidence of RCT increases in patients with comorbidities that promote this dysfunction [hypertension, diabetes mellitus (5), obesity (6)].

Several authors (20) have shown that patients with RCT exhibit a state of hypoxia that contributes to the pathogenetic process. A study by Benson et al. (21) highlighted an increase in the Hif-1a factor (produced in hypoxic environments) in patients with a supraspinatus tendon tear.

Hypoxia also represents an important stimulus for the expression of ET-1 mRNA (22).

In a study conducted on smooth muscle cells of pulmonary arteries, it was also observed that ET-1 itself can induce hypoxia and, consequently, promote an increase in the concentration of Hif-1a both through increased synthesis and reduced degradation (23). Therefore, a potential positive feedback loop between the increased concentration of ET-1 and hypoxia can be hypothesized.

Several studies have demonstrated how ET-1 can prove to be a useful marker in the early diagnosis and in determining the severity and prognosis of a large variety of pathologies, such as acute heart failure (24), septic shock (25), bronchopulmonary dysplasia (26), acute pancreatitis (27), interstitial lung disease (28), Parkinson disease (29).

A study by Kalles et al. (30) demonstrated that in some pathological conditions it is not ET-1 that is increased, but its precursor, Big ET-1, which further studies of this type should take into consideration to analyze, possibly comparing the values of ET-1 and Big ET-1 in cases and controls. However, it is also true that on the contrary, in other conditions such as arterial hypertension, it is ET-1 that is increased and not Big ET-1 (31).

In addition to the diagnostic and prognostic utility of ET-1, ET-1 receptor antagonists have been studied as a possible therapeutic approach given their ability to act on the hemodynamic component (32-35).

For these reasons we believe that it could be interesting to delve deeper into the topic, therefore further studies are needed. Currently, there are no studies in the literature investigating the plasma concentration and the role of ET-1 in patients with RCTs.

We observed no statistically significant difference between the case group and the control group. However, there are few hypothesis that could explain our results.

In our cases, RCTs are a pathological condition resulting from a chronic degenerative process, with those one due to trauma excluded. Probably, the cuff tendon stumps have undergone reparative fibrosis, thus losing the endothelial component that would have led to ET-1 synthesis.

Approximately half of the cases examined also have one or more ongoing medical conditions under treatment. It has been demonstrated that drugs such as angiotensin-converting enzyme (ACE) inhibitors and statins can inhibit the gene expression of ET-1 (16), resulting in reduction of ET-1 plasma levels. These factors may explain the lack of a statistically significant difference between the case and control groups in our study.

Furthermore, various studies have demonstrated that NO production occurs during chronic tendinopathy (36-38), with NO being an inhibitor of the synthesis of ET-10.

Another regulator of ET-1 is the ET-B receptor, which acts as an antagonist to the ET-A receptor, promoting vasodilation by stimulating the synthesis of NO and prostacyclins, factors that can reduce the expression of the ET-1 gene. It also increases the clearance of ET-1 and facilitates the reuptake of ET-1 by endothelial cells (16). Immunohistochemistry studies could be useful for locating and quantifying these receptors at the site of the lesion.

We did not observe a statistically significant difference when comparing ET-1 values in relation to the severity of the tear. Moreover, the average value is higher in small tears compared to large and massive tears, although this data could be affected by non-uniform sample distribution among the three groups.

The present study has some limitations that need to be considered. It is a case-control study and so could not completely resolve issues concerning temporality. Future studies on the topic could be organized as prospective cohort studies, to overcome this limitation.

The duration of potential risk factors for RCT were not available. However, there is no evidence to suggest that both the case and control groups were different enough to account for the risk estimates found in this study. Thus, while some confounding is possible, it appears unlikely to account for these results.

The small sample is the major limitation; however, considering the cost of the procedure, these preliminary results may open a line of research on undocumented functions of ET-1.

Another limitation is represented by the population of controls, which may not be a representative sample of the general population, because it is represented only by the population of hospital employees enrolled in the same facility where surgery on cases was performed.

Conclusions

As of today, there are no similar studies in literature, so this study should be interpreted as a starting point. From the preliminary results obtained, no statistically significant differences between patients with RCT and healthy controls were found regarding plasma ET-1 concentration. Furthermore, ET-1 seems to not play a role also in the severity of RCT.

Considering the current limitations of the study and the preliminary data obtained, further studies will be needed to confirm or not the role of ET-1 in the pathogenesis and severity of RCT. This will require increasing the sample size, improving the selection of cases and controls, planning a prospective cohort study, and expanding the research to include other analytes, such as ET-1 precursors.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://aoj.amegroups.com/article/view/10.21037/aoj-24-24/rc

Data Sharing Statement: Available at https://aoj.amegroups.com/article/view/10.21037/aoj-24-24/dss

Peer Review File: Available at https://aoj.amegroups.com/article/view/10.21037/aoj-24-24/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aoj.amegroups.com/article/view/10.21037/aoj-24-24/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of Sapienza University of Rome (No. 32/2021) and written informed consent was obtained from all patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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