Dissertation committee:
Ιωάννα Ανδρεάδου
Καθηγήτρια Φαρμακολογίας, Τμήμα Φαρμακευτικής, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Ευστάθιος Καστρίτης
Καθηγητής Θεραπευτικής –Παθολογίας/Ογκολογίας , Ιατρική Σχολή, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Μελέτιος Αθανάσιος Δημόπουλος
Πρύτανης Εθνικού και Καποδιστριακού Πανεπιστημίου Αθηνών, Καθηγητής Αιματολογίας-Ογκολογίας, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Κωνσταντίνος E. Βοργιάς
Καθηγητής Bιοχημείας, Τμήμα Βιολογίας Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Ιωάννης Πιρμεττής
Ερευνητής Α και Διευθυντής Ερευνών στο Ινστιτούτο Πυρηνικών και Ραδιολογικών Επιστημών, Τεχνολογίας, Ενέργειας και Ασφάλειας του ΕΚΕΦΕ «Δημόκριτος
Εμμανουήλ Μικρός
Καθηγητής Φαρμακευτικής Χημείας, Τμήμα Φαρμακευτικής, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Ανδρέας Παπαπετρόπουλος
Καθηγητής Φαρμακολογίας, Τμήμα Φαρμακευτικής, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Background and introduction: The term amyloidosis encompasses a wide spectrum of diseases characterized by protein misfolding and the deposition of amyloid fibrils in various tissues. Light chain amyloidosis (AL) is a plasma cell dyscrasia in which clonal plasma cells overproduce amyloid-forming immunoglobulin light chains, and cardiac involvement of the disease is associated with inferior survival. Treatment of AL amyloidosis relies on chemotherapeutic approaches aimed at eliminating the plasma cell clone. The prognosis of AL is based on the evaluation of cardiac markers such as natriuretic peptides or troponin levels even if the amyloid is not localized in the cardiac tissue, but to date there are no specific treatments aimed at reducing myocardial damage. In fact, standard treatments for heart failure are ineffective or intolerable in AL with cardiac involvement. The complexity in AL is based on the fact that on the one hand amyloid disrupts the tissue architecture and at the same time, the circulating light chains induce cardiotoxicity. Today, the underlying mechanisms of cardiotoxicity have not been elucidated and therefore we have not concluded on druggable targets for cardioprotection and improvement of the cardiac dysfunction.
Purpose: The aim of the thesis is to compare the cardiotoxicity of light chains derived from patients with AL amyloidosis and cardiac involvement in relation to other plasma cell dyscrasias such as multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) without cardiac complications and to improve our understanding of the mechanisms of induced cardiac damage in AL. More specifically, the objectives are 1) gene sequencing and biotechnological production of full-length light chains from patients with AL and cardiac involvement, from MM, MGUS patients or non-clonal light chains from healthy volunteers (HV), 2) the investigation of the light chain induced cardiotoxicity in three distinct cell populations, 3) the investigation of toxicity mechanisms both in clinical samples and in vitro 4) the creation of an in vivo murine model of amyloidogenic light chain cardiotoxicity and 5) the establishment of an AL amyloid model suitable for the study of molecules targeting the amyloid.
Methods: CD138+ clonal plasma cells were isolated from bone marrow from patients with AL and cardiac involvement (n=8), with MM (n=2) and with MGUS (n=2) and peripheral blood mononuclear cells were isolated from HV (n=2) to isolate RNA and characterize the light chain gene family repertoire. The genes encoding the clonal light chains and three light chains from HV were cloned and expressed in Shuffle E.coli cells. Light chain folding, oligomerization, and their propensity to form amyloids were assessed by circular dichroism spectroscopy, electrophoresis and electron microscopy, respectively. Primary adult murine ventricular cardiomyocytes (pAVMCs), primary fibroblasts from the myocardium and primary smooth muscle cells from aorta were exposed to various concentrations of light chains to assess cell death and investigate the mechanisms of cardiotoxicity. In parallel, circulating markers were determined in the plasma of AL patients and proteomic analysis on patient derive fat aspirates was performed to reveal potential biomarkers and molecular mechanisms associated with the tissue damage. To establish an in vivo light-chain induced cardiotoxicity model, C57BL6 male mice were randomized into 6 groups and subjected to intramyocardial administration of the three cardiotoxic and amyloidogenic light chains, the isotypic light chains from HV, or the vehicle. We characterized the model via echocardiography ultrasound evaluation of cardiac function, histological evaluation and we studied the mechanisms of the induced toxicity. Balb/c mice were randomized and injected subcutaneously with amyloid-rich solutions prepared from amyloidogenic peptides or full-length light chains of the patients to establish the AL amyloidoma model. Three molecules that are flat, lipophilic and can bind to Alzheimer's disease amyloid deposits were used as candidate radiodiagnostics to characterize the model using single photon emission tomography (SPECT) imaging and biodistribution assays.
Results: We successfully produced and isolated 7 full-length light chains from AL patients, 2 from MM, 2 from MGUS and 3 from HV. All light chains were similar in conformation as a beta-sheets and in their oligomerization as a mixture of monomers and dimers. Four light chains derived from patients with AL and cardiac involvement led to cardiotoxicity in pAVMCs that was associated with their amyloid-forming property, and three of them also induced toxicity in vascular smooth muscle cells. Light chains derived from HV, MM and MGUS did not exhibit cytotoxicity. Proteomic analysis in adipose tissue of AL amyloidosis patients revealed that signaling pathways related to endoplasmic reticulum (ER) and inflammation are important. The corresponding toxicity mechanisms were also identified in vitro in pAVMCs since incubation with amyloidogenic and toxic light chains increased ER stress markers such as Bip and IRE-1a proteins which lead to increased apoptosis in kappa light chains and to increased autophagy in lambda type chains. Kappa light chains from AL, MM, and MGUS patients led to increased inflammation, suggesting that this mechanism is independent of the observed toxicity. In the circulation of patients with AL amyloidosis, an increase in cyclic guanosine monophosphate (cGMP) was detected, which was associated with an increase in the flow-mediated dilatation (FMD). This increase was also significant in patients who have AL amyloidosis with cardiac involvement and was also related to levels of cGMP release from vascular smooth muscle cells. The cytotoxic light chains led to an increase in ER stress in vascular smooth muscle cells as well. Thus, in both cell populations the use of the IRE-1a inhibitor prevented light chain-induced cytotoxicity. In vivo, intramyocardial administration of the three cytotoxic and amyloidogenic light chains resulted in cardiotoxicity with obvious histological alterations. The kappa light chain exhibited cardiotoxicity with a characteristic phenotype of cardiomyocyte apoptosis and preserved systolic function while the lambda chains led to reduced systolic function, interstitial fibrosis and increased autophagy. A common mechanism of toxicity of the administered light chains was shown to be the increase in ER stress markers. The AL amyloidoma model induced by full-length light chain amyloids from patients with AL amyloidosis was found suitable for the characterization of amyloid-binding molecules, and through its use we identified one of the three candidate radiodiagnostics as the best for detecting AL amyloids in vivo.
Conclusions: Light chains deriving from patients with AL amyloidosis and cardiac involvement induce cardiotoxicity, which correlates with their amyloidogenic potential, and ER stress is an important pathway for the development of cardioprotective factors. We developed two animal models, and their use is expected to contribute to the development of cardioprotective agents and pharmacomolecules targeting the amyloid deposits.
