A transcriptomic study of Hereditary Angioedema attacks

Background: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. Objective: We sought to explore the involvement of other putative genes in edema formation. Methods: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. Results: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. Conclusions: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE

Hereditary angioedema: Assessing the hypothesis for underlying autonomic dysfunction

Background Attacks of Hereditary Angioedema due to C1-inhibitor deficiency (Cl-INH-HAE) are often triggered by stressful events/hormonal changes. Objective Our study evaluates the relationship between autonomic nervous system (ANS) and contact/complement system activation. Methods Twenty-three HAE patients (6 males, mean age 47.5 +/- 11.4 years) during remission and 24 healthy controls (8 males, mean age 45.3 +/- 10.6 years) were studied. ECG, beat-by-beat blood pressure, respiratory activity were continuously recorded during rest (10') and 75degrees- head-up tilt (10'). C1-INH, C4, cleaved high molecular weight kininogen (cHK) were assessed; in 16 patients and 11 controls plasma catecholamines were also evaluated. Spectral analysis of heart rate variability allowed extraction of low-(LF) and high-(HF)frequency components, markers of sympathetic and vagal modulation respectively. Results HAE patients showed higher mean systolic arterial pressure (SAP) than controls during both rest and tilt. Tilt induced a significant increase in SAP and its variability only in controls. Although sympathetic modulation (LFnu) increased significantly with tilt in both groups, LF/HF ratio, index of sympathovagal balance, increased significantly only in controls. At rest HAE patients showed higher noradrenaline values (301.4 +/- 132.9 pg/ml vs 210.5 +/- 89.6 pg/ml, p = 0.05). Moreover, in patients tilt was associated with a significant increase in cHK, marker of contact system activation (49.5 +/- 7.5% after T vs 47.1 +/- 7.8% at R, p = 0.01). Conclusions Our data are consistent with altered ANS modulation in HAE patients, i.e. increased sympathetic activation at rest and blunted response to orthostatic challenge. Tilt test-induced increased HK cleavage suggests a link between stress and bradykinin production

Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency

BackgroundHereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A2 (sPLA2s).ObjectiveWe sought to investigate the enzymatic activity and biological functions of sPLA2 in patients with C1-INH-HAE.MethodssPLA2s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA2 (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA2 indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.ResultsPlasma sPLA2 activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA2 activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.ConclusionsPLA2 enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA2s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets

Deciphering the genetics of primary angioedema with normal levels of C1 inhibitor

The genetic alteration underlying the great majority of primary angioedema with normal C1 inhibitor (nl-C1-INH-HAE) cases remains unknown. To search for variants associated with nl-C1-INH-HAE, we genotyped 133 unrelated nl-C1-INH-HAE patients using a custom next-generation sequencing platform targeting 55 genes possibly involved in angioedema pathogenesis. Patients already diagnosed with F12 alterations as well as those with histaminergic acquired angioedema were excluded. A variant pathogenicity curation strategy was followed, including a comparison of the results with those of genotyping 169 patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE), and only filtered-in variants were studied further. Among the examined nl-C1-INH-HAE patients, carriers of neither the ANGPT1 p.Ala119Ser nor the KNG1 p.Met379Lys variant were found, whereas the PLG p.Lys330Glu was detected in four (3%) unrelated probands (one homozygote). In total, 182 different variants were curated, 21 of which represented novel mutations. Although the frequency of variants per gene was comparable between nl-C1-INH-HAE and C1-INH-HAE, variants of the KNG1 and XPNPEP1 genes were detected only in nl-C1-INH-HAE patients (six and three, respectively). Twenty-seven filtered variants in 23 different genes were detected in nl-C1-INH-HAE more than once, whereas 69/133 nl-C1-INH-HAE patients had compound heterozygotes of filtered variants located in the same or different genes. Pedigree analysis was performed where feasible. Our results indicate the role that alterations in some genes, like KNG1, may play in disease pathogenesis, the complex trait that is possibly underlying in some cases, and the existence of hitherto unrecognized disease endotypes

Immune response covaries with corticosterone plasma levels under experimentally stressful conditions in nestling barn swallows (Hirundo rustica)

Traits related to fitness are often pleiotropically linked or otherwise constrained in their expression. Organisms therefore trade between fitness components such as number and viability of their offspring. The physiological mechanisms mediating such trade-offs, however, have been poorly investigated. We manipulated brood size and satiation of nestling barn swallows, Hirundo rustica, to simulate the effect of two kinds of natural stresses, i.e., long-term intense competition in a large brood and acute food deprivation, and we measured their effect on body condition, T cell-mediated immune response, and corticosterone, the main hormone mediating the adrenocortical stress-response. Brood enlargement increased corticosterone levels compared with those for brood reduction, and brood enlargement depressed immune response, body mass, and condition. Corticosterone levels markedly increased after food deprivation. Immune response negatively covaried with corticosterone levels measured after long-term stress. Hence, living in a crowded nest and with food deprivation elicited a stress response mediated by corticosterone, and depressed an important component of offspring fitness such as T cell-mediated immunity. The negative covariation between circulating corticosterone and immunity suggests that the trade-off between offspring number and quality is mediated by variation in plasma levels of corticosterone, which has immunosuppressive effects

A case of remittent C1-inhibitor deficiency

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and kinin/contactsystem activation. Mutations in C1-inhibitor gene almost consistently result in reduced C1-inhibitor functional level in plasma causing hereditary angioedema, a life-threatening autosomal dominant disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlyingth is variability remains undisclosed. We report a case of a patient suffering from abdominal pain and presenting markedly reduced C1-inhibitor plasma levels, episodically undergoing spontaneous normalization, car- rying the Arg378Cys missense mutation in the serpin domain. Immunostaining analysis of patient plasma revealed the presence of C1-inhibitor oligomers, together with the occurrence of a SDS stable band that disappeared in reducing conditions, suitable for a disulphide bridged Arg378Cys homodimer. Expression studies in eukaryotic cell lines resulted in a drop in mutant C1-innhibitor secretion compared to wild type and confirmed the plasma observations. Notwithstanding, the purified recombinant proteins behave similarly. Both proteins formed stable covalent complexes with target proteases, and the kinetic of inhibition of the mutant was just slightly diminished, although this reduction increased with temperature.Thus,our findings suggest that the Arg378Cys C1-inhibitor mutant once correctly folded should maintain the wild type functional and structural features and instead it should bear a folding defect, abnormally susceptible to environmental factors, which may occasionally promote protein oligomerization. Moreover it can form a disulphide linked homodimer. Both these processes could account for its variability in plasma levels

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress

Updates on Novel Non-Replacement Drugs for Hemophilia

Over the last decade, the world of hemophilia has experienced an unprecedented therapeutic advance, thanks to the progress in bioengineering technologies, leading to the introduction of drugs with novel mechanisms of action based on restoring thrombin generation or coagulation factor VIII mimicking. Apart from the bispecific monoclonal antibody emicizumab, already approved for patients with severe hemophilia A with and without inhibitors, novel non-replacement drugs designed to reduce the treatment burden of patients with hemophilia A or B with or without inhibitors are undergoing evaluation in clinical trials. Thanks to their innovative mechanism of action and subcutaneous administration, these drugs promise to provide effective bleeding protection together with improved adherence and improve health-related quality of life for patients with hemophilia. On the other hand, rare thromboembolic events have been reported with some of these drugs and warrant continuous post-marketing surveillance and investigation of predisposing factors, although the overall safety profile of most of these drugs is good. Finally, new challenges need to be faced in the clinical and laboratory monitoring of the hemostatic status in patients treated with these innovative therapies. In this review, we provide an update on the available data on novel non-replacement drugs currently undergoing evaluation in clinical trials for patients with hemophilia