ABSTRACT
Background: Rheumatoid arthritis (RA) is the most common systemic autoimmune disease that primarily affects joints but is also often characterized by extra-articular involvement1. Cardiovascular diseases are the most important causes of sudden death in these patients, which present a risk of developing cardiovascular events increased by 48%2. The causes of increased cardiovascular risk are several and not completely understood, but recent evidence supports the key role of endothelial dysfunction in pathogenesis. In this complex scenario, it is known that IL-6 receptors are present at the endothelial level and can be activated leading to endothelial dysfunction. SARS-Cov-2 is a coronavirus responsible for the disease called 'coronavirus disease 2019' (CoViD-19) characterized by clinical manifestations ranging from a flu-like syndrome up to severe lung damage associated with systemic hyper cytokine syndrome that can lead to multiple organ failure and death. Therefore, both RA and Covid-19 are associated with an increased pro-thrombotic and cardiovascular risk and IL-6 might be crucial in the patho-physiological mechanisms of both diseases. Objectives: The main hypothesis of this study was to evaluate the possible role of IL-6 as a promoter of endothelial dysfunction in RA and CoViD-19. Methods: In vitro experiments were conducted on the endothelial cell line EA. hy926. Cells were treated for 24 h with fetal bovine serum (FBS), a pool of RA patients' sera or a pool of CoViD-19 patients' sera. The expression levels of adhesion molecules (V-CAM1/CD-106, I-CAM/CD-54, p-selectine/CD-62, tissue factor/CD-142) and apoptosis were analyzed using cytofuorimetric technique. In addition, the autophagy level, using the autophagy markers p62 and LC3II, were evaluated through a western-blot analysis. The same experiments were conducted co-treating cells with the same pool of sera in addition to tocilizumab (TCZ), an anti-IL-6 drug, to verify the reversibility of the process and test the role of the aforementioned cytokine. Data are reported as interquartile median values. The Kruskal Wallis test was used for unpaired samples and the Mann-Whitney test for paired samples. P<0.05 values were considered statistically signifcant. Results: EA. hy926 cells, when treated with both RA and CoViD-19 patients' sera, showed increased levels of activation molecules and apoptosis compared to FBS treated cells. In addition, we observed increased levels of both p62 and LC3 proteins after both rheumatoid arthritis and CoViD-19 patients' sera treatment. All these fndings were reversible in the presence of TCZ. The results are presented in Figure 1. Conclusion: Our data showed that treatment with RA and CoViD-19 patients' sera increase the activation and death of endothelial cells in vitro. The increased level of cells death is possibly due to a block of autophagy. The reversibility of the process after blocking IL-6 with TCZ co-treatment confrms the hypothesis that IL-6 can play a key role in the pathogenesis of endothelial damage in patients with RA and CoViD-19.
ABSTRACT
Myocardial infarction is a global health burden for which there is no treatment available that aims to recover the damaged tissue after the ischemic event. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in their application for mRNA-based covid-19 vaccines. After myocardial infarction, endogenous mechanisms that enable repair of the functional damaged tissue can be triggered by modified mRNA (modRNA) delivery, locally in the infarcted area. As a first step, in order to optimize the LNP formulation for effective myocardial delivery and study cellular tropism of the LNPs in the heart, different LNPs formulations will be evaluated as delivery systems in a murine healthy heart model. Different LNP formulations varying in type and amount of helper lipid were used as delivery systems for modRNA encoding the reporter genes luciferase or eGFP. In vitro, LNPs were evaluated for modRNA delivery in a human endothelial cell line (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, modRNA delivery was evaluated in C57BL-6 mice, undergoing open chest heart surgery under general anaesthesia in order to infuse LNPs into the left ventricular wall. For determination of luciferase expression levels, animals were infused with luciferin substrate intraperitoneally 24 hrs after injection. Heart, liver, lungs, spleen and kidneys were extracted for imaging in a bioluminescence imaging system. The organs were then stored in liquid nitrogen for further ex-vivo modRNA delivery analysis. For determining cellular tropism, histology was performed on mice treated with eGFP modRNA. Both bioluminescence imaging and luminescence analysis in tissue lysates showed that mRNA transfection is achieved in the myocardium 24 hours after LNP intramyocardial administration. However, all LNP formulations also resulted in high expression levels in other organs, including liver and spleen. Changes in type or amount of helper lipid in LNPs strongly affected transfection levels. Histology of the treated hearts revealed a distinct transfection pattern. The targeted, interstitial cells were negative for CD31 (marker for endothelial cells and monocytes) and Troponin I3 (marker for cardiomyocytes) (Figure 1). We show that, using an optimized LNP formulation, a significant degree of modRNA local transfection of the heart can be achieved. However, despite the local route of administration (into the left ventricular wall), the highest LNP transfection is shown in remote organs such as liver and spleen. More improvements of the LNP formulations must be done to increase their tropism towards the heart tissue for their optimization as cardiac delivery systems. Determining which cell types are being targeted is also important in order to establish a therapeutic target when applying the LNPs for cardiac therapy. (Figure Presented).