Investigating the effects of proteasome inhibition on endothelial cells

Abstract

Multiple myeloma is a genetically complex condition that affects plasma cells. Innovative treatment methods are rapidly emerging that improve clinical outcomes. Clinical success has been demonstrated with proteasome inhibitors that target the mechanism of cellular protein homeostasis. The Food and Drug Administration approved Carfilzomib (CFZ), a second-generation proteasome inhibitor, to treat multiple myeloma patients who have relapsed or are resistant to other treatment options. It inhibits the chymotrypsin-like activity of the 20S immunoproteasome from binding to the β5 subunits irreversibly. Proteasomes serve as important degradation mechanisms within the Endoplasmic reticulum (ER) that maintain cellular homeostasis. When the proteasome is compromised, chronic proteotoxic stress within the endoplasmic reticulum results, ultimately resulting in multiple myeloma cells' death. CFZ has a highly effective profile accompanied by low renal toxicity; despite this, clinical studies have linked the proteasome inhibitor to an increase in cardiovascular adverse events. Several studies have described cardiotoxicity caused by CFZ, with an endothelial component possible. However, CFZ has not yet been studied concerning endothelial dysfunction. This in vitro study aims to investigate the impact of CFZ on endothelium homeostasis in the potential adverse cardiovascular effects. This was accomplished by incubating human vein endothelial cells (HUVECs) with CFZ at a clinically relevant dose for one hour and sometimes with TNFα/IL-1β individually or combined following CFZ treatment. This was followed by detecting gene and protein expression of relevant markers of ER stress, inflammation, and thrombosis, and later, some functional studies were conducted at various time points after incubation. The data gathered from this study showed that CFZ increased ubiquitin expression up to 24 hours post-CFZ treatment in both sub-confluence and confluence HUVECs. However, while there was increased expression of BiP, the principal ER stress marker in sub-confluence cells indicating induction of ER stress, resting HUVECs were not affected by CFZ on the expression of BiP and other ER stress markers, suggesting the CFZ cannot cause ER stress in resting HUVECs.Interestingly, although CFZ had no consequence on the expression of procoagulant and pro-inflammatory endothelial cells markers, it enhanced the expression of some of the well-known endothelial-derived anticoagulants mediators, thrombomodulin (TM), endothelial nitric oxide enzyme (eNOS), phosphorylated eNOS (P-eNOS), tissue plasminogen activator (t-PA), and Krüppel-like factor 4 (KLF4). Moreover, CFZ was able to diminish TNFα mediated TF, VWF, and ICAM-1 expression and promoted TM, eNOS, and KLF-4 upon TNFα treatment, suggesting a protective effect on the endothelium. Consistent with this, CFZ decreased clot formation and factor Xa (FXa) generation on the endothelium after TNFα treatment and, likewise, reduced platelet capture, leukocyte adhesion, and VWF string formation on HUVECs induced by TNFα. I also explored the role of eNOS in preserving ordinary ER statues and as an essential regulator of CFZ cytoprotective effect on HUVECs, while BiP did not exhibit the same role in these cells. In conclusion, the data obtained from this study revealed that CFZ is incapable of provoking ER stress in confluence resting ECs and attenuates the pro-thrombotic and pro-inflammatory effects of TNFα on the endothelium, thus displaying a potential cytoprotective profile on the endothelium.