Functional Autoantibodies in ST-Elevation Myocardial Infarction (FAST study): Investigating in vitro the Role of Agonistic Autoantibodies Targeting Receptors in Human Cardiac Microvascular Dysfunction

Background: Coronary microvascular obstruction (CMVO) is a severe form of microvascular dysfunction and the pathophysiological substrate of the no-reflow phenomenon, a major prognostic factor in revascularized ST-elevation myocardial infarction (STEMI). Emerging clinical evidence from our group identified agonistic autoantibodies targeting angiotensin II receptor type 1 (AT1R-AAs) and endothelin-1 receptor type A (ETAR-AAs) as associated with CMVO. To explore the specific immune-mediated cellular effects in the hearts of STEMI patients, a novel in vitro model of microvascular dysfunction was generated to replicate in vitro the exposure of human cardiac microvascular endothelial cells (hcMVECs) to these AAs. Methods: hcMVECs were stimulated with individual IgG preparations from STEMI patients with high titers of AT1R-AAs and ETAR-AAs (n=4). Healthy subjects with under-threshold titers served as controls (n=4). Receptor activation specificity was verified by co-administering the antagonists valsartan and bosentan. Cellular effects were assessed before (T0) and post-treatment, at 24 (T24) and 48 hours (T48). Cell morphology was evaluated via bright-field microscopy and immunofluorescence for VE-cadherin and F-actin. VCAM-1 expression served as a marker of endothelial activation. Mitochondrial superoxide detection was utilized to measure oxidative stress. Any variations in cell proliferation and cytotoxicity were quantified using colorimetric assays. AT1R and ETAR distribution in hcMVECs was also investigated by immunofluorescence. Results: AAs-positive STEMI IgGs induced morphological changes in hcMVECs, which appeared smaller and displayed cytoskeletal disarray. Conversely, treatment with receptor antagonists promoted cell elongation and realignment. Nuclear morphometric analysis revealed a significant time-dependent increase in circularity in the presence of valsartan and bosentan, with both STEMI and control IgGs. At T24, hcMVEC activation was markedly higher upon stimulation with STEMI IgGs, compared to control IgGs. At the same time point, hcMVECs exposed to STEMI IgGs exhibited also an increased mitochondrial superoxide production, which was mitigated by receptor blockade. Cell proliferation was higher than baseline in all treatment conditions, with a clear decrease over time (except for cells exposed to both control IgGs and receptor inhibitors). Receptor blockade reduced cytotoxicity, as significantly observed at T48. Lastly, the typically diffuse pattern of AT1R and ETAR likely assumed a more pronounced perinuclear localization in STEMI IgGs-treated cells, an effect partially reversed by the drugs. Conclusions: Our in vitro modeling findings suggest that AT1R-AAs- and ETAR-AAs-positive IgGs from STEMI patients induce structural alterations, activation, and oxidative stress in hcMVECs through specific receptors-mediated action. This cardiac microvascular endothelial model has the potential to further elucidate the mechanism of action of these functional autoantibodies at the basis of clinically observed STEMI-associated CMVO and develop personalized treatment strategies for patients with high titers.