Synergistic Effect of Carfilzomib and Metformin in Vascular Plasticity; The Emerging Role of Autophagy

Introduction: Carfilzomib (Cfz) correlates with a risk of reversible cardiotoxicity in 5-10% of multiple myeloma (MM) patients. We have recently shown that metformin (Met) has a prophylactic role against the Cfz-induced cardiotoxicity in vivo, through activation of AMPKα signaling (Blood 2019;133:710-23). However, the impact of Cfz on vascular function is obscure. Therefore, we sought to investigate: i) the acute, ii) the sub-chronic effect of Cfz on the vascular reactivity, iii) the effect of metformin co-administration on the vascular phenotype and iv) the impact of Cfz and Met co-administration on aged Human Aortic Smooth Muscle Cells (HAoSMCs).

Methods: Forty male C57Bl/6 mice were assigned as follows: Acute Protocol: i. Normal Saline (N/S 0.9%); ii. Cfz (8 mg/kg); iii. Met (140 mg/kg); iv. Cfz+Met (8 mg/kg; 140 mg/kg respectively). N/S and Cfz were injected intraperitoneally (IP), while Met was administered via oral gavage daily, for 2 days (n=5 per group). Mice were sacrificed 48h after the initiation of treatments. Sub-chronic Protocol: i. N/S 0.9%; ii. Cfz (8 mg/kg); iii. Met (140mg/kg); iv. Cfz+Met (8 mg/kg; 140mg/kg respectively) for 6 days. N/S and Cfz was injected IP, while Met was administered via oral gavage on alternate days for 6 days (n=5 per group). Mice were sacrificed on the 7^th day after the initiation of treatments. At the endpoint of the experiments (48h and 7 days respectively), heparinized whole blood (WB) samples and aortic tissues were collected from the experimental animals. WB samples were used to determine leucocytes derived ROS production (oxidative burst) and aorta sections (3 mm) underwent ex vivo vascular studies (relaxants: Achetlyocholine (ACh), nitroglycerin (Gtn) contractant: PDGF2α). The remaining aortic tissue underwent molecular Western blot analysis for the elucidation of the underlying mechanism. For the in vitro experiments HAoSMCs Passage 7-8 were subjected to Cfz (0.1, 0.3μM), Met (10μM, 10mM) treatment and to the combination of the two compounds. Subsequently, cells were treated with the optimal concentrations of the compounds 24h after treatment with Angiotensin II (AngII, 100nM), CoCL₂ (150μM) and 48h after Glucose (25μM) to simulate the hypertensive, hypoxic and diabetic stimuli respectively. Proliferation was assessed by MTT; LC3IIB (as a marker of autophagy) expression was analyzed by immunofluorescence (IF) and cellular bioenergetics and ATP production were investigated through Seahorse XF analyzer, facilitating the study of intracellular glycolysis and oxidative phosphorylation.

Results: In the acute setting, Met did not ameliorate the ROS production induced by Cfz, while no effect of Cfz on vascular relaxation/contraction was evident. In the sub-chronic setting, Met limited WB macrophage-derived oxidative burst and, while Cfz did not affect vascular reactivity, the co-administration of Cfz and Met increased PDGF2α induced aortic contractility, deducing an increased plasticity of the vessel. Western blot analysis of the aortas revealed that Cfz led to a de-phosphorylation of AMPKα, an upregulation of mTOR and iNOS while induced endoplasmic reticulum (ER) stress as indicated by increased Calnexin, IRE1a and Bip expression. In the Cfz+Met group, Met restored AMPKα phosphorylation and partially abrogated ER stress markers calnexin and Bip. However, the co-administration of the agents led to a synergistic amplification of autophagy as shown by LC3IIB expression. In the aged HAoSMCs, only Met exhibited cytotoxic capacity, while Cfz failed to exhibit any cytotoxicity. Moreover, Met was found to totally abrogate mitochondrial oxidative phosphorylation and shift cellular ATP production to anaerobic glycolysis, while Cfz had no effect on cellular bioenergetics. In terms of cardiovascular risk factors, the co-treatment of Cfz and Met led to an additive cytotoxic effect in presence of CoCL₂ and AngII. Finally, in all tested conditions Cfz and Met were found to have a synergistic effect in inducing autophagy, as shown by LC3IIB upregulation.

Conclusions: Sub-chronic co-administration of CFz and Met increases vascular plasticity in vivo, through a converging AMPKα- and ER stress-dependent autophagy. In vitro, Cfz does not present any effect on aged cells, probably due to the decreased proteasome activity, while Met synergizes with Cfz to decrease proliferation through alterations in cellular bioenergetics and induction of autophagy.