HAE-C1INH patients were consecutively enrolled during ambulatory visits from June 2023 to April 2024 at Istituti Clinici Scientifici Maugeri, IRCCS, Milan, Italy. The inclusion criteria were: i) confirmed diagnosis of HAE-C1INH according to guidelines (5); ii) age between 18 and 65 years; iii) ability to provide a written informed consent. The exclusion criteria were: i) any type of chronic diseases requiring chronic treatment (i.e. hypertension, previous myocardial infarction, diabetes, chronic heart failure, autoimmune disease, neurodegenerative diseases); ii) active acute diseases; iii) Sars-Cov2 infection in the previous 3 months (23); iv) acute HAE attacks in the 7 days preceding the experimental procedure; v) acute HAE attacks in the 72 hours after the study participation (a posteriori exclusion). Age- and gender- matched HC were recruited between healthcare professionals and their relatives. Non-HAE-related inclusion and exclusion criteria were applied to select the HC group.

The sample size was calculated over the systolic arterial blood pressure variations from REST phase to SECPT (see experimental protocol below) based on data derived from Schwabe et al. (16). Assuming 80% power, a two-sided α of 0.05, and a 1:1 allocation between HAE-C1INH patients and HC, the sample size calculation indicated a minimum of 17 participants per group.

The experimental protocol was approved by the Ethics Committee of Istituiti Clinici Scientifici Maugeri (approval number 2774 CE; date of approval 31 May 2023) and was registered on ClinicalTrials.gov (NCT06414252). The study was compliant with the Declaration of Helsinki principles and each subject signed a written informed consent.

An overview of the experimental protocol is provided in Figure 1.

All sessions were conducted between 9:00 and 12:00 a.m. in a quiet room with a comfortable temperature to minimize circadian variability of all the registered variables. All participants were instructed to refrain from caffeine or exercise before testing. All the participants underwent a medical visit to verify the inclusion and exclusion criteria and to collect demographic and clinical data.

Continuous data were presented as mean ± standard deviation, while categorical variables as absolute number and percentage. Continuous demographic and clinical characteristics of HC and HAE-C1INH patients were compared by Student t test in case of normal distribution or Mann-Whitney test in case of non-normal distribution. χ2 test was applied for categorical variables. Two-way repeated measures analysis of variance (ANOVA, Holm-Sidak test for multiple comparisons) was performed to verify the differences of AP and HR parameters, psychological variables and levels of inflammatory cytokines between the two groups at the different time points. A p<0.05 was considered significant for all the performed analyses. The statistical analyses were conducted with Sigmaplot software (Systat Software, Inc., Chicago, IL, version 11.0).

Twenty HAE-C1INH patients (9 males, mean age 44 ± 14 years) and 20 HC individuals (9 males, mean age 45 ± 15 years) were enrolled in the study. The demographic and clinical features are presented in Table 1. Nine HAE-C1INH patients experienced attacks in the previous 6 months, 2 had one attack, 4 more than two, and 3 more than three; 18 patients were on long-term prophylaxis (LTP): 15 with lanadelumab, and 3 with subcutaneous C1 esterase inhibitor. Compared to HC, patients had a higher body mass index.

In the HC group, two participants did not complete the 3-minute SECPT (a 38-year-old man: 70 s; a 63-year-old woman: 74 s). In the HAE-C1INH patient group, five participants failed to complete the test (a 26-year-old woman: 73 s; a 33-year-old man: 42 s; a 37-year-old woman: 50 s; a 66-year-old woman: 65 s; a 58-year-old woman: 96 s).

The comparison between results obtained in the psychological questionnaires in patients and HC is presented in Table 2. Patients obtained higher scores in the HADS anxiety, in the global score of PCS and its magnification domain. Non-significant differences in the HADS depression and PSS were found between the two groups.

The results of the HR, SAP and DAP mean values during REST, SECPT, POST10 and POST40 are reported in Figure 2. HR, SAP and DAP increased from REST to SECPT in both patients and HC and decreased in both groups during POST10 and POST40 when compared to SECPT. HR, SAP and DAP values were similar in REST, POST10 and POST40. HR was lower in patients than HC in all the experimental phases.

The results of the inflammatory cytokines quantification are presented in Table 3 and summarized in Figure 3. Cytokines quantification was performed in 19 patients and 18 HC. Compared to HC, patients were characterized by higher levels of IL-6 and TNF-α in all the experimental phases (with the exception of IL-6 in POST40). The values of IL-1ß, IL-6 and TNF-α did not vary across the different phases in both groups.

The results of the perceived level of stress in patients and HC are reported in Figure 4. The perceived level of stress during REST was equal in both patients and HC, but was higher in patients during SECPT (60.6 ± 34.3 vs 34.6 ± 23.8, p<0.05).

The perceived level of stress increased from REST to SECPT in both patients and HC. In addition, patients and HC reported similar level of unpleasantness (57.9 ± 31.4 vs 52.0 ± 30.0, respectively) and pain (59.5 ± 32.2 vs 50.7 ± 29.5) at the end of the SECPT phase as measured by the VAS scales.

Patients exhibited a cardiovascular response to the SECPT comparable to that of HC, suggesting that the disease does not appear to compromise their ability to cope with this specific stimulus during attack-free periods.

This finding is noteworthy, as increasing evidence in recent years has indicated the involvement of the ANS in HAE-C1INH (28, 29). Indeed, previous studies have documented that, during the early phase of an impending attack, patients display lower HR compared to the attack and post-attack phase, suggesting that an ANS impairment intervenes during the prodromal phase. The present study evaluates a different setting and demonstrates that their ability to respond to an acute stressor, such as the one induced by the SECPT remains preserved.

Specifically, patients showed increases in HR as well as in SAP and DAP during the 3-minutes cold-water hand immersion, compared to baseline values, and returned to resting levels during the 40-minutes recovery period, mirroring the responses observed in HC.

In healthy individuals, increases in SAP and DAP during the SECPT have been consistently reported in numerous studies (30–33). Conversely, the expected HR response to hand immersion remains more controversial. Not all studies have described a stable and significant HR increase across participants (30).

While some investigations reported a sustained HR elevation (33), others observed an initial marked increase followed by a gradual decline (32). Such discrepancies may be attributable to the complex and not yet fully understood mechanisms underlying ANS regulation during the SECPT. The SECPT is known to activate the sympathetic nervous system, inducing vasoconstriction (34). The individual response to the SECPT depends on both the integrity of the sympathetic noradrenergic efferent neurons and afferent baroreflex (35) pathways, and may also be modulated by individual differences in pain receptor activation (30). In the present study, the use of mean HR and AP values does not allow for definitive conclusions regarding the contribution of each underlying mechanism. Nevertheless, the key observation remains that patients and HC did not differ in their HR and AP responses to the SECPT.

A growing number of studies reported that acute psychological laboratory stressor tests increase the concentration of the inflammatory mediators (36) and that there is an interrelationship between inflammatory biomarkers and pain (37). However, previous research on cytokine responses specifically to the SECPT remains insufficient. Our results showed that the considered inflammatory cytokines did not change across the different experimental phases either in patients or HC. The lack of detectable cytokine changes in our study can be due to several factors, such as the 40 minutes post-stress time-point being too early to observe measurable increases in plasma, with changes occurring primarily at the cellular level rather than in circulation during this period. Additionally, the cytokine response to the SECPT may have been subtle or short-lived, falling below the detection threshold or resolving before the time of measurement. Among the limited number of studies investigating cytokine responses to the SECPT, one involving healthy individuals found that those with a high subjective perception of stress showed elevated salivary IL-1β levels immediately before the SECPT (38). Since salivary cytokines may reflect local rather than systemic inflammation, we opted for plasma measurements to ensure a more reliable assessment of circulating inflammatory activity. A more recent report involving 53 potentially traumatized women and age-matched HC found significantly elevated plasma levels of IL-6, 90 minutes post-SECPT relative to baseline levels collected 15 minutes before the stressor (39) suggesting that detecting cytokine changes may require a later post-SECPT blood sampling time-point.

An important finding of our study is the increased levels of TNF- α and IL-6 in patients compared to HC, indicating an augmented inflammatory state in patients in resting condition. Notably, TNF- α and IL-1 plasma levels were found to be significantly elevated in HAE-C1INH patients during attacks and also in remission in comparison to HC in a previous study (40). In partial agreement with our data, Gramstad and colleagues (41) documented an elevated baseline thrombo-inflammatory load in HAE patients in remission, characterized by significantly higher concentrations of several pro-inflammatory cytokines, including IL1β, IL-6, TNF- α, compared to HC (41). It has to be noted that patients had a higher BMI than HC, which could also be a stimulus for chronic inflammation. Collectively, these data support the hypothesis that a chronic, low-grade inflammatory state may persist in HAE-C1INH patients even beyond the acute phase of the disease.

Our results showed that patients reported significantly higher levels of anxiety symptoms than HC. This finding is consistent with previous studies conducted on similar patient populations (42). Moreover, patients exhibited higher scores on the magnification subscale of the PCS, indicating a stronger tendency to overestimate or exaggerate the perceived threat and severity of pain. Such results are expected given the chronic, debilitating, and sometimes painful nature of HAE (43), as well as the well-documented relationship between pain experience and catastrophizing in both acute and chronic conditions (44). Nonetheless, although this difference reached statistical significance, the mean PCS values in both groups remained below the clinically relevant cut-off (45), suggesting that the observed increase in pain magnification is subclinical and does not necessarily translate into heightened pain perception.

Regarding perceived stress, both patients and HC showed an increase from baseline following the SECPT, confirming that the procedure effectively induced stress in both groups. However, patients reported significantly higher perceived stress than HC immediately after the SECPT. This finding parallels the elevated pain magnification observed in the same group and aligns with prior evidence showing that magnification tendencies are positively associated with perceived stress (46). Taken together with the elevated baseline inflammatory markers observed in our study, these results further support the established link between psychological stress and inflammatory activity, which may contribute to the onset or exacerbation of angioedema attacks.

Beyond these differences in perceived stress and pain magnification, the psychological profile of patients may reflect altered stress appraisal. The SECPT combines physical discomfort with a social-evaluative threat, known to elicit stronger affective responses in individuals characterized by heightened anxiety. The higher HADS-anxiety and pain magnification scores observed in patients, although subclinical, could indicate a cognitive bias toward threat interpretation, amplifying the perception of stress even in the absence of exaggerated physiological activation. Additionally, maladaptive attentional focus on bodily sensations has been shown to reinforce sympathetic arousal and inflammatory signal through activation of the HPA axis and the SAM system (47, 48). This mechanism could partly explain the coexistence of elevated perceived stress and increased baseline IL-6 and TNF-α levels observed in these patients. Future studies should explore whether interventions targeting stress appraisal and pain catastrophizing, such as mindfulness-based or cognitive-behavioral approaches, could reduce both psychological distress and inflammatory activation, ultimately mitigating stress-related disease triggers in these patients.

None of the patients experienced attacks within 72 hours following the test, although a significant difference in perceived stress immediately after the SECPT was detected when compared with HC. Several interpretations may account for this observation. First, it is possible that the procedure was not sufficiently intense or personally meaningful to induce a physiological response capable of triggering an attack. Second, LTP could have played a protective role, influencing the occurrence of attacks. Finally, it cannot be excluded that acute stress does not constitute an effective trigger for attacks in this patient population. Further studies are needed to disentangle these possibilities and to clarify the mechanisms underlying stress responsiveness in this context. Moreover, the potential contribution of chronic stress exposure to attack occurrence remains an open question deserving future investigation.

Although our experimental design was complex and methodologically rigorous, the limited sample size restricts the generalizability of our findings, which should therefore be replicated in larger cohorts. Additionally, nearly all patients included were undergoing LTP treatment, whose effects on inflammatory markers and stress reactivity remain unknown. Between protocol definition and start of data collection the pharmacological scenario of LTP changed drastically: at the moment of enrolment most of the subjects who agreed to participate were receiving LTP. Unfortunately, the sample size of the present study does not allow to test the contribution of LTP. These factors should be carefully considered in future research to better isolate the impact of stress reactivity on disease expression.

Lastly, the observed difference in BMI between the groups was unforeseen and difficult to interpret unequivocally in relation to its impact on HR. Nonetheless, BMI values in both groups fall within or are close to the normal range for Western populations. Even so, future research should consider including BMI among the matching criteria for selecting control subjects, along with age and sex.