Hereditary angioedema (HAE) is a rare genetic condition characterized by unpredictable episodes of subcutaneous or submucosal swelling attacks due to increased vascular permeability, secondary to the release of bradykinin (1–4). HAE can manifest with angioedema (AE) attacks in different locations, including the extremities, genitals, face, and gastrointestinal tract, as well as the upper airway, where they can be potentially life-threatening (1, 2, 4, 5).

HAE is divided into two types, HAE with C1 esterase inhibitor deficiency (C1INH) named HAE-C1INH, caused by mutations in the SERPING1 gene, coding for C1INH and HAE without C1INH deficiency, known as HAE with normal C1INH (HAE-nC1INH) (1). HAE-C1INH is categorized into two subtypes: type 1, characterized by a quantitative deficiency of C1INH, and type 2, marked by a qualitative deficiency of C1INH. In HAE-nC1INH, several mutations have been described in genes coding proteins targeting the kallikrein-kinin system, such as factor XII (6, 7), plasminogen (8) or kininogen (9) and the vascular endothelium, such as angiopoietin (10), myoferlin (11) and heparan sulfate-glucosamine 3-O-sulfotransferase 6 (12). HAE-nC1INH due to mutations in the F12 gene coding coagulation factor XII, is known as HAE-FXII and is the most frequent cause of HAE-nC1INH. Furthermore, in some patients no mutation has been described and are considered as HAE of unknown mechanism (HAE-UNK) (1).

HAE attacks initiate upon local activation of the contact system (CS) involving the interaction of the kallikrein kinin system (KKS), the fibrinolytic system and the coagulation system. FXII is activated (FXIIa) when it contacts with a negatively charged surface or macromolecules (1–5, 13). FXIIa cleaves the prekallikrein (PK) and the high molecular weight kininogen (HMWK) complex, which subsequently turns into plasma kallikrein (pK), and bradykinin (BK) is released (1–5). FXIIa and pK can additionally activate FXII in a positive feedback-loop, leading to an amplified response (2, 4, 5, 9, 13, 14).

BK is a vasoactive nonapeptide that acts on the surface of endothelial cells by binding to its B2 (mainly) and B1 receptors increasing vascular permeability, and subsequently resulting in AE attacks (2–5, 13, 14). BK is rapidly metabolized by several enzymes, such as angiotensin-converting enzyme (kininase II, ACE), carboxypeptidase N (kininase I), aminopeptidase P (APP), neutral endopeptidase (NEP), and dipeptidyl peptidase IV (DPPIV) (13–15).

Acute phase reactants (APR) are serum proteins that exhibit significant increases in various inflammatory conditions, whether acute or chronic (16–18). Commonly utilized APR include erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum amyloid A (SAA), which play crucial roles in the diagnosis and monitoring of numerous diseases such as severe bacterial or viral infections, as well as chronic inflammatory disorders like Crohn’s disease and rheumatoid arthritis (16–20). Additionally, in certain conditions, elevated levels of other APR such as haptoglobin, alpha-1-antitrypsin, C3, C4, hepcidin, or fibrinogen may also be observed (16–18). D-dimer is generated through the breakdown of the fibrin mesh by plasmin, an enzyme responsible for fibrinolysis. Additionally, D-dimer acts as an APR, triggering elevated levels of cytokines such as IL-6. Consequently, this reciprocal interaction suggests that D-dimer and other fibrin degradation products might impact inflammatory and acute-phase responses by fostering neutrophil and monocyte activation, thus stimulating the release of IL-6 (21–23).

When an HAE attack occurs, a local increase of BK has been described (13), however the role of inflammation in HAE was been poorly explored. Therefore, the aim of this study was to investigate the involvement of inflammatory mediators in patients with HAE during the acute attacks.

This study shows, that conventional inflammatory pathways may be involved in the pathophysiology of acute AE attacks in HAE. Despite the limited acute sample size, we have demonstrated a substantial increase of some APRs such as SAA, ESR and D-dimer during AE attacks, both in HAE-C1INH and in HAE-FXII patients.

Coagulation parameters and markers of activation of the fibrinolytic pathway, such as D-dimer and the contact system had been previously explored in HAE-C1INH (21, 24, 25). In agreement with findings from previous studies (21, 24, 25), we have observed an increase of D-dimer during attacks in HAE-C1INH patients. Noteworthy, this study shows for the first time that AE attacks can also increase D-dimer in HAE-FXII. D-dimer is produced during the breakdown of the fibrin mesh by plasmin. Elevation of D-dimer in HAE-C1INH attacks may mean secondary hyperfibrinolysis, rather than hypercoagulation, because plasminogen levels during baseline periods did not differ from those recorded during AE attacks and are similar to the healthy control levels (24). In a study of 79 patients with HAE-C1INH, 80% of them presented an increase in D-dimer during AE attacks, that was higher in patients with angioedema affecting the submucosa (abdominal and oropharyngeal-laryngeal) compared to subcutaneous (peripheral and facial) (25). Nevertheless, severity and activity of HAE-C1INH was not considered. In our study, apart from abdominal attacks, we have also observed an increase of D-dimer during facial and peripheral attacks. However, these differences should be evaluated cautiously and could be related to the small sample size. In our study, D-dimer was elevated in symptom-free periods only in 14.5% of patients. One study showed a significant elevation of D-dimer at baseline compared with controls (24). This discrepancy can be due to the different study design, since we have considered an elevation of D-dimer when values were over the normal threshold, but we have not compared patients with controls. In addition to fibrinolysis contributing to the generation of D-dimer in HAE patients via contact system activation, it is crucial to underscore that inflammatory states trigger the production of fibrinogen in response to interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor (TNF) (18). This reciprocal interaction suggests that D-dimer and other fibrin degradation products might impact inflammatory and acute-phase responses by promoting neutrophil and monocyte activation, thus facilitating the release of IL-6 (22, 23). In HAE attacks, an elevation of IL-6 has been demonstrated (26), so it is plausible to hypothesize that the activation of the inflammatory cascade and the production of fibrinogen by endothelial and inflammatory cells in HAE patients can contribute to the production of D-dimer (18). Nevertheless, additional research is required to validate this hypothesis.

APRs are known to respond to pro-inflammatory cytokines, such as IL-1, IL-6 or tumor necrosis factor-alpha (TNF-α), and to the local production of endothelial nitric oxide (NO), prostaglandins, and endothelial adhesion molecules (16–18). Although the liver is the main producer of APR, other non-hepatic sources such as endothelial cells and macrophages have been identified (18–20). SAA, which belongs to the apolipoprotein family, is produced mainly in the liver and its secretion reasonably increases during inflammatory states. In vitro IL-6 has a synergism in the production of SAA either in combination with IL-1β or with TNF-z (27). SAA is present constitutively in plasma, and the increase in SAA levels observed in our patients during AE attacks could be in response to the local production of proinflammatory cytokines (IL-6, IL1-β and TNF-z) independently of the hepatic production, as proposed in Figure 2 .

ESR, is an indirect measure of the amount of fibrinogen produced during inflammatory states, such as infections, in malignancy or other immune diseases (16–18).

We have noted a rise in ESR among a subset of our patients during symptom-free intervals. Notably, during acute attacks, a substantial proportion of our patients experienced a significant increase in ESR, indicating the occurrence of systemic inflammation. Until now, no prior study had investigated the role of ESR during HAE acute episodes. While we did not measure fibrinogen levels, a plausible explanation for our observations could be that, following the activation of the contact system and fibrinolysis, there is a localized production of fibrinogen during attacks. In angioedema due to ACE inhibitors (ACEi-AE), a type of AE that, like HAE-C1INH and HAE-FXII, is mediated by BK, a 1.5-fold increase in fibrinogen has been observed during attacks (28). Moreover, fibrinogen is able to increase the vasodilator potency of BK by 10-fold and to increase bradykinin-induced vasodilator-stimulated phosphoprotein phosphorylation (28). This potentiation of bradykinin-induced vasodilation suggest that fibrinogen might contribute to the pathophysiology of ACEi-AE. An elevation of CRP has also been described in ACEi-AE (29–31). CRP, is another APR, mainly produced in the liver, widely used as a biomarker in inflammatory diseases, that increases mostly in response to microbial components during infections and in autoimmune diseases (18, 32–34). CRP primarily responds to IL-6 and, to a lesser extent, to IL-1β and TNF-α (18, 33). In accordance with a previous study in 25 HAE-C1INH patients (35), we found that CRP levels were within normal range during baseline and remained unaltered during HAE attacks. On the other hand, during HAE attacks, levels of IL-1, IL-6 and TGF-b have been found to be significantly higher compared to remission and those from healthy controls, whereas no activation of liver CPR production has been observed (26). Furthermore, it has been established that CRP may be produced extrahepatically by various tissues, including neurons, adipose tissue, lungs, kidneys, and intestines (33, 34). In fact, in our study, CPR remained unaltered even in abdominal attacks, so further studies are needed to elucidate the reason for the lack of CRP production during AE attacks in HAE patients compared to ACEi-AE (29).

We postulate that in individuals with HAE, the heightened levels of SAA, ESR and D-dimer during acute attacks could be a result of the localized production of proinflammatory cytokines. Specifically, SAA and ESR may be increasing locally in response to the release of IL-6, IL-1β, and TNF-α, rather than systemic proinflammatory cytokines, suggesting a limited hepatic APR production. This discrepancy might explain the absence of CRP, which is primarily produced in the liver. Moreover, various immune cells, including eosinophils, basophils, neutrophils, mast cells, and lymphocytes, express BK1 and BK2 receptors on their surfaces (36). This cellular composition suggests a possible pivotal role in the inflammatory process during HAE attacks, as these cells could be activated through BK (5). Consequently, in patients with HAE, bradykinin may be implicated in the local production of APR. Figure 2 illustrates how the release of bradykinin during HAE attacks may contribute to acute phase reactant production through immune cell activation and subsequent cytokine release, highlighting the complex interplay between inflammation and the contact system in HAE pathophysiology.

Reliable biomarkers for distinguishing between various types of HAE are currently scarce, with genetic studies being a primary means of differentiation. Assessment of C4 levels and C1INH activity and function remains crucial in delineating HAE types (1, 2). Furthermore, there is a notable gap in potential biomarkers for discerning between different types of angioedema (e.g., mast cell-mediated or bradykinin-induced) (1, 2). Bradykinin has been proposed as a potential biomarker, but its rapid metabolism and the technical challenges associated with its measurement make it impractical for routine use (37–39). Other groups have put forth alternative candidates. The potential use of HMWK and the plasma PK-HK complex has been explored (37), although standardization of techniques for their quantitation remains a hurdle, and they have not yet been incorporated into routine clinical practice. Elevated levels of adrenomedullin and urokinase plasminogen activator (uPAR) during acute attacks, measured using transcriptomics, have also been suggested as potential biomarkers, although these techniques require further standardization before widespread use (40). Finally, Tie-2 (Angiopoietin-1 receptor), fibroblast activation protein-alpha (FAP-α), and tissue plasminogen activator (tPA) have also been postulated as biomarkers to distinguish between different types of AE (41). However, it is crucial to acknowledge that none of these biomarkers have yet been incorporated into routine clinical practice, and their measurement requires complex techniques. SAA, ESR, and D-dimer, being easily measurable, could be regarded as for AE biomarkers. Nevertheless, a comprehensive analysis is required to understand the role of APR in other types of angioedema. High SAA levels were found in 67 patients with chronic spontaneous urticaria (CSU), with a particularly significant elevation in those with both CSU and AE (42). Furthermore, in anaphylaxis, mast cell activation has been linked to contact system activation (43, 44), and in CSU, an increase in plasma D-dimer has been reported, correlating with disease activity (45–47). Although still an unexplored area, it is conceivable that in mast cell-mediated angioedema, APR could also see an increase during acute episodes.

To our knowledge, this study shows for the first time that SAA and ESR are elevated in HAE-C1INH and in HAE-FXII attacks and D-dimer is elevated also in HAE-FXII attacks, since the elevation in AE attacks in patients with C1INH deficiency has been previously described. Clearly, large scale follow-up studies are needed to elucidate the behavior and clinical usefulness of SAA and ESR in different HAE types.

In conclusion, our study provides valuable insights into the inflammatory mechanisms involved during AE attacks in HAE patients. While these attacks are localized, the significant elevation of APRs suggests a broader inflammatory response. The causal relationship between increased APR levels and HAE attacks warrants further investigation.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Comité d’Ètica. Hospital Universitari Vall d’Hebron. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

JG-S: Writing – review & editing, Writing – original draft, Visualization, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation. ML-H: Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization. PG-B: Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis. AS-C: Writing – original draft, Writing – review & editing, Project administration, Methodology. PB: Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis. JP-G: Writing – original draft, Writing – review & editing, Project administration, Investigation, Formal analysis. OL: Writing – original draft, Writing – review & editing, Validation, Supervision, Methodology. VC: Writing – original draft, Writing – review & editing, Visualization, Validation, Supervision, Methodology. MG: Data curation, Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.