Impact of inhibitors of Angiotensin II axis and NAD metabolic booster on inflammatory profile of peripheral blood mononuclear cells from COVID-19 patients with heart failure – ANICOV-HF
Impact of inhibitors of Angiotensin II axis and NAD metabolic booster on inflammatory profile of peripheral blood mononuclear cells from COVID-19 patients with heart failure
SARS-COV2 (COVID-19) was shown to increase Angiotensin II peptide hormone. Angiotensin converting enzyme inhibitors (ACEIs) and receptor blockers (ARBs) are prescribed drugs in cardiovascular disease that could be crucial players in COVID-19 prognosis, owing that ACE2 is one SARS-CoV2 binding site and that ACE2 expression is markedly elevated following ACEIs/ARBs treatment. In parallel, studies have shown that infected celles require more NAD coenzyme to defend against SARS-COV2 infection.
Our goals are to assess whether i) ACEIs/ARBs could reduce the inflammatory response associated with COVID-19 rather than worsening it, ii) the anti-inflammatory potential of NAD boosters
ACE2 is known to be protective by opposing the action of ACE and converting ANGII to ANG(1,7) which acts as a vasodilatator. . Evidence suggests that ACEIs/ARBs could also exert protective effects in SARS-COV2 patients through reversing the significant increase in ANGII following SARS-COV2 infection by converting it into the protective ANG (1, 7) and through limiting excessive activation of the immune system.<br />In agreement, some studies showed that hypertensive and heart failure SARS-COV2 patients who are on ACEIs/ARBs, showed a decrease in the plasma pro-inflammatory cytokine levels of both IL-6 and Th1/Th2 ratio. Accumulating evidence directly links the severity of SARS-CoV2 infection to CRS (cytokine release syndrome). The cytokine storm observed with SARS-CoV2 infection is directly related to the overactivation of an uncontrolled immune responses of peripheral blood mononuclear PBMCs subsets resulting in severe acute respiratory. PBMCs are well known to express the RAS components and there is a general consensus that RAS components of PBMCS are involved in modulating inflammatory response depending on the state of balance in which they reside. With dominating ACE/ANGII/AT1R activity, PBMCS exert a pro-inflammatory profile, whereas a dominating ACE2/ANG1-7/AT2 activity promotes an anti-inflammatory state [19-22]. Mitigating the induction of circulating inflammatory responses by blocking AT1 receptors on immune cells by ACEIs/ARBs has been documented . For instance, ACEIs are long known for their direct impact on the immune system with their ability to suppress PBMCs from releasing pro-inflammatory cytokines including IL-1 and TNFalpha, two major cytokines involved in SARS-CoV2 induced CRS.<br /><br />Cellular response to SARS-CoV2 infection: The NAD-PBMC signaling hypothesis<br />Nicotinamide Adenine Dinucleotide (NAD) is a major coenzyme for energy metabolism and a signaling molecule notably involved in the regulation of inflammatory response. Indeed, replenishment of NAD level through nicotinamide riboside (NR) precursor treatment reduces mitochondrial ROS (mtROS) production and enhances mitochondrial respiratory in PBMCS, while reducing the production of pro-inflammatory markers in healthy elderly subjects and heart failure patients. SARS-CoV-2 infected tissues strikingly upregulate several poly (ADPribose) polymerases (PARPs) that consume large quantities of NAD to perform their role in the antiviral response and repair of ROS-induced DNA injuries. Based on strong preliminary and published evidence we hypothesize that stages A-C heart failure SARS-CoV2 patients on ACEIs/ARBs exhibit beneficial cardiovascular clinical outcomes during the symptomatic phase with a better prognosis at three months post-recovery. Mechanistically, we hypothesize that the observed protection with ACEIs/ARBs is due in part to the modulation of PBMCs inflammatory profile, which could be further boosted by NR treatment.
This is a longitudinal study divided into two independent but highly integrated and complementary arms: 1) a clinical arm investigating the clinical impact of SARS-COV2 infection in patients with different stages (A-C) of heart failure in the presence or absence of ACEIs/ARBs at admission and at three months post recovery, and 2) a molecular arm that performs an in-depth analysis of the inflammatory profile, injury profile, and PBMCs profile in blood samples acquired from all the recruited patients in this study at admission and three months post recovery and assess their response to NR. The aims proposed in this study will be thoroughly investigated by experts in cardiovascular research between France and Lebanon. The clinical arm will be mainly investigated at AUBMC in Lebanon and the molecular arm will be mainly investigated at Paris-Saclay in France. Blood (25 mL) will be collected from all the patients and healthy donors recruited in this study during the acute phase of infection and during the three-months follow up visit. Collected blood will be divided into one sample processed for immediate blood work at Dr. Zouein’s Lab and a second sample for PBMCs isolation and cryopreservation to be shipped to Dr. Mericskay’s Lab. PBMCs will be isolated using the state-of-the-art Sepmate technique developed by Stem Cells Technologies (STI) and cryopreserved using STI detailed protocol using Cryostor CS10 at minimum of 5 to 10x 105 Cells/mL. Of note, from healthy blood, PBMCs yield ranges between 0.5 to 3 x 106 cells per mL blood for 10 mL blood. We anticipate this yield to be much higher in SARS-COV2 infected patients. PBMCS will be identified by flow cytometry as CD66b-CD45+. The Sepmate isolation technique generates intact PBMCs that are compatible with all the proposed experiments. Variables: Dr. Zouein’s Lab will investigate the inflammatory profile, Ang II/Ang(1-7) levels, NAD levels (whole blood), and neutrophils to lymphocytes ratio (NLR) using EISA kits, LC-MS and other techniques as previously described [32, 45]. Dr. Mericskay’s lab will thoroughly investigate PBMCs for NAD+/NADH levels by biochemical assays and for the expression profile of 48 genes related to NAD metabolism, nicotinamide, nicotinamide mononucleotide, etc.) by LC-MS and for the expression profile of 60 genes related to NAD metabolism (NAMPT, NMRK1/2, PARP1-16, SIRT1-7, etc.) and inflammation (NLRP3, IFNa/b, IL1b, IL6, IL18, etc.) and RAS system expression using a custom-designed TaqMan Gene Expression Array Card.
At the intermediate stage of the project, the results are the following. The first part of the project was to dedicated to the completion of the first clinical study initiated by Dr Zouein medical team in AUB. They recruited 176 patients with COVID-19 infection and cardiovascular comorbidities in the American University of Beirut Medical Center in Lebanon. 110 patients were taking ACEI or ARB and 66 were not. They collected the data and looked at inflammatory markers such as CRP and IL-6 and cardiac markers such as Troponin and compared the results between the two groups of patients. They reported the incidence of Acute respiratory distress syndrome (ARDS), sepsis and death of each patient and compared the 2 groups. We found that patients taking ACEI and ARB had a statistically significant decrease in levels of Troponin, IL-6 and CRP compared to patients not taking these medications (p< 0.05). We found no difference in rates of ARDS, sepsis, or death between the 2 groups.
Conclusion: The inhibition of the Renin-Angiotensin-Aldosterone-System had no effect on the mortality of patients with COVID-19 and on their overall disease progression. It may also be beneficial not to stop these medications as they decrease the inflammation in the body and the levels of troponin which are related to increased stress on the heart.
The second part of the project is dedicated to a molecular arm that performs an in-depth analysis of the inflammatory profile, injury profile, and peripheral blood mononuclear cells (PBMCs) profile in blood samples acquired from recruited patients. New patients needed to be recruited after the stard of the financial agreement between the participating French and Lebanese institutions. At this intermediate stage of the projet, the recruitment is still ongoing.
At this intermediate stage, one article is already in preparation describing the outcome of the first clinical arm of the project.
The SARS-CoV-2 (COVID-19) outbreak emerged in Wuhan, China in late 2019, and rapidly spread throughout the world, resulting in an ongoing pandemic with a serious global health burden. To date, France reported 5728090 confirmed SARS-COV2 cases and 105850 deaths while Lebanon reported 531243 confirmed SARS-COV2 cases and a total of 7415 deaths that are rising on daily basis. Recommendations are to maintain SARS-COV2 surveillance since a resurgence in contagion remain possible until 2024. SARS-COV2 manifestation is ranging from asymptomatic infections to severe acute respiratory distress syndrome (ARDS) and serious cardiovascular complications and high risk of death. Multiple emerging studies confirmed that SARS-COV2 infection is associated with an increase in a vasoactive compound known as angiotensin II (ANGII) which is associated with adverse cardiovascular outcomes in predisposed patients, such as heart failure patients. In addition, ANGII signaling has been shown to exacerbate the inflammatory response but also to interfere with the energy metabolism and signaling, notably through the Nicotinamide Adenine Dinucleotide (NAD) coenzyme.
Angiotensin converting enzyme inhibitors (ACEIs) and ANGII receptor blockers (ARBs) prescribed drugs could be a crucial player in SARS-COV2 prognosis, owing that ACE2 is one of SARS-CoV2 binding sites and that ACE2 expression in the cardiovascular system is markedly elevated following the treatment with ACEIs and ARBs. ACE2 is known to be protective by opposing the action of ACE and converting ANGII to ANG (1,7). Any ACE-ACE2 activity imbalance, defines whether or not the injury in response to a stimulus will occur and in the context of SARS-COV2 infection, data from patients indicated a decreased ACE2/ACE ratio. Multiple recent studies associated ACEIs and ARBs with reduced risk of SARS-COV2 disease after adjusting to a wide range of variables. These findings support preliminary data generated from 66 SARS-COV2 hospitalized patients with stage A-C heart failure at the hospital of the American University of Beirut, demonstrating that ACEIs and ARBs treatment decrease the risk of death, ventilation requirement, and troponin levels as a marker of myocardial injury, suggesting a potential protective effects with potential improved prognosis (IRB approved protocol).
Therefore, we hypothesize that stages A-C heart failure SARS-CoV2 Lebanese patients on ACEIs/ARBs exhibit beneficial cardiovascular clinical outcomes during the symptomatic phase with a better prognosis at three months post-recovery. There is a direct pro- link between ANGII signaling and inflammation detected in peripheral blood mononuclear immune cells (PBMCs). NAD is a major coenzyme for energy metabolism and a signaling molecule notably involved in the regulation of inflammatory response and shown to oppose ANGII deleterious effects on vascular and cardiac cells as well as macrophages. Therefore, we also hypothesize that the observed protection with ACEIs/ARBs is due in part to the modulation of PBMCs inflammatory profile by preventing their excessive activation and their subsequent induction of the cytokine storm syndrome (CRS) that is associated with a very poor prognosis in SARS-COV2 patients. NAD signaling pathway boosters such us nicotinamide riboside (Vitamin B3) will be used on PBMCS isolated from SARS-COV2 patients to test the importance of NAD signaling in modulating PBMCs inflammatory profile as an adjunct to ACEIs/ARBs therapy. It could contribute to reduce mitochondrial ROS (mtROS) production and enhance mitochondrial respiratory in PBMCS, hence toning down an aggressive immune response. All the investigators involved in this study combine a strong expertise in clinical cardiovascular management of heart failure patients, basic cardiovascular research in inflammation and NAD pathways, SARS-COV2 patients recruitment, and clinical and basic biostatistical analysis.
Monsieur Mathias MERICSKAY (Signalisation et physiopathologie cardiovasculaire)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CARPAT Signalisation et physiopathologie cardiovasculaire
AUBMC Heart Repair Laboratory of the Department of Pharmacology and Toxicology, Medical Center,American University of Beirut
Help of the ANR 94,998 euros
Beginning and duration of the scientific project: January 2022 - 18 Months