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Potential therapeutic targets for COVID-19 complicated with pulmonary hypertension: a bioinformatics and early validation study – Scientific Reports

After the initial observation by Xie and colleagues12 that COVID-19 is associated with an increase in cardiovascular disorders, numerous clinical studies and meta-analyses have confirmed an augmented incidence of acute coronary syndromes, myocarditis, pericarditis, heart failure, and arrhythmias13,14,15,16,17,18,19,20 The consequences of severe COVID-19 include systemic hypoxia, acute respiratory distress, hyper-coagulation, sepsis, inflammation, metabolic stress, and cytokine storms, all of which may stress the cardiovascular system, eventually leading to blood pressure dysregulation21,22,23,24,25.

Pulmonary hypertension, as a serious cardiopulmonary complication of COVID-19,increasing the likelihood of requiring intensive care unit care, mechanical ventilation, extracorporeal membrane oxygenation (ECMO), and even death. Therefore, detecting high pulmonary artery pressure in SARS-CoV-2 patients early might enhance the long-term prognosis of patients and minimize the hospitalization rate and death owing to such complications7,8,9,26.But de Jong CMM27 found that chronic thromboembolic pulmonary hypertension is not a more common long-term complication after COVID-19-associated pulmonary embolism than after non-COVID-19-associated pulmonary embolism. Whether this phenomenon is due to large differences in pathogenesis, it is particularly important to study the pathogenesis of COVID-19 combined with pulmonary hypertension.

So, this study attempted to explore molecules associated with the pathogenesis of COVID combined with PH through a variety of bioinformatics methods, after which a comprehensive diagnostic pattern was established by scoring significant markers. The expression of each gene was quantified and scored, with higher scores associated with greater predictive potential. These predictive scores could be used for monitoring and early intervention in COVID-19 patients, especially those with PH. A further aim was the identification of specific drugs that could target key genes associated with COVID-19 complicated with PH, enhancing both the diagnosis and treatment of this condition.

Similar to the genetic causes of pulmonary arterial hypertension, COVID-19 severity may be affected by variations in the same genes. Many of the pathobiological hallmarks of pulmonary arterial hypertension are also present in COVID-19-induced pulmonary vasculopathy, including endodermatitis, vasculitis-medial hypertrophy, and smooth muscle cell proliferation. Especially endothelial dysfunction is a common feature of the clinical manifestations observed in COVID-19 patients. The SARS-CoV-2 coronavirus accesses host cells via the binding of its spike glycoprotein to angiotensin-converting enzyme 2(ACE2), sialic acid receptor, transmembrane serine protease 2 (TMPRSS2), and extracellular matrix metalloproteinase inducer (CD147); catepsin B and L also participate in virus entry. All of these factors are expressed in endothelial cells28,29,30,31. Overactivated platelets cause cytokine storms and thrombosis, and studies have shown that platelets that express pro-inflammatory molecules and that carry viral RNA are particularly likely to be highly active32.

Among the C-DEGs, we found 47 highly expressed genes and 15 lowly expressed genes when comparing COVID-19 and PH datasets. Using GO analysis, we found that C-DEGs were enriched for the following terms: inflammatory response, immunological response, cell surface receptor, cellular response to interferon-gamma, positive regulation of NF-kB, and reaction to lipopolysaccharide. TNF signaling route, Herpes simplex virus 1 infection, viral protein interaction with cytokine and cytokine receptor, malaria, lipid and atherosclerosis, PPAR signaling pathway, and PPAR signaling pathway were all enriched for among the C-DEGs in the KEGG enrichment analysis. These results strongly suggest that immune inflammation is a driving force in the emergence and progression of COVID-19 coupled with PH. Infection with SARS-CoV-2 may cause a cytokine storm leading to systemic inflammation and vascular endothelial cell damage. These changes may cause hypercoagulability and intravascular thrombosis, together with increasing pulmonary vascular resistance. Diffuse microangiopathy and microthrombosis caused by extensive impairment of vascular endothelial function may further aggravate the imbalance of the pulmonary ventilation/blood flow ratio, and increases in the pulmonary right-to-left shunt may further aggravate hypoxia, thus promoting pulmonary vascular contraction and remodeling. In addition, when the body is attacked by a virus, it can activate T cells and promote the overexpression of IFN-induced genes that could also lead to the apoptosis of endothelial cells and thus to PH. This has much in common with the results of LuisG’s study33.

To further search for the core genes associated with COVID-19 combined with PH among the 62 identified DEGs, we used machine learning to choose potential diagnostic biomarkers. LASSO regression analysis identified 20 genes with the lowest binominal deviation. The random forest approach identified 10 candidates after ranking the DEGs according to significance, and the SVM-RFE method identified 8 genes with the lowest error and best accuracy after 100 fold changes. Using the intersection of the results of the three algorithms, SELE and CCL20 were identified as core genes. These two key genes were discovered to have statistically significant differences when compared to one another in the validation set. Next, we built a prediction model to make even more informed predictions on COVID-19 complicated with PH by assessment of the scores in the table in which higher scores represented greater likelihood of developing PH after COVID-19. By drawing the ROC, PR, and DCA curves, it was found that the prediction model was reliable.

One of the core genes identified was SELE. Activation of endothelial cells by cytokines results in the expression of a cell-surface glycoprotein called SELE, which facilitates the adherence of circulating monocytes and lymphocytes to endothelial cells34. The plasma marker for endothelial dysfunction or injury is soluble E-selectin (sE-selectin), which is secreted by damaged or dysfunctional endothelial cells35,36. IFN-gamma-R2 membrane transport to the Golgi and proper IFN-gamma-R assembly both need E-selectin involvement. The activation of the BTK kinase is triggered by the interaction of an E-selective protein, which in turn forms a functional IFN-gamma-R that can bind to another functional IFN-gamma-R, so generating an efficient innate response of macrophages to intracellular bacterial infection37,38. The importance of endothelial cells in the spread of SARS-CoV-2 is becoming more widely accepted. It is probable that E-Selectin’s function in leukocyte chemotaxis during inflammation is the fundamental mechanism at work here. E-Selectin surface expression increases, which may facilitate the entry of leukocytes into the tissue and the initiation of inflammation to combat the infection39,40.DM Smadja et al. discovered that PH is linked to circulating endothelial cells, soluble E-selectin, and sVCAM, but not to endothelial progenitor cells, CD34(+)CD133(+) cells, or vascular endothelial growth factor (VEGF)41. Similarities between this and our study’s results suggest that SELE may be a key pathogenic molecule of COVID 19 coupled PH.

CCL20, also known as macrophage inflammatory protein-3α or liver activation regulated chemokine, is another gene of central importance that we examined. CCL20 is a CC chemokine that specifically interacts to CCR6. In addition to recruiting immature dendritic cells, effector/memory T cells, and B cells, this chemokine also has an inflammatory role in maintaining homeostasis. It plays a crucial part in maintaining regular trafficking of immune cells and in kicking off T-cell-dependent inflammation. CCL20 regulates the right amount of inflammation by keeping a fine balance between offensive and defensive immunity42,43,44,45. It also recruits Th17 cells and regulatory T cells to inflammatory sites since CCR6 is present on these cell types. Patients with COVID-19 were found to have elevated levels of CCL20 in both bronchoalveolar lavage (BAL) fluid and plasma samples46. Pulmonary arterial hypertension in individuals with SSc is associated with elevated serum CCL20 levels44. However, there has only been a little amount of research done on CCL20 in COVID and PH patients.

Functional enrichment was primarily in adaptive immune response, leukocyte, and lymphocyte mediated the immune response, and proinflammatory response mediated by cytokines like IL-12 and TNF-a, which promoted the proliferation of pulmonary artery smooth muscle cells and induced vascular remodeling, as determined by GSEA of SELE and CCL20 in the data sets of COVID-19 and PH.

We used ssGSEA analysis to look at the relationship between SELE and CCL20 and 23 different types of immune cells in the COVID-19 and PH datasets, and we found that activated CD4 T cells, activated dendritic cells, natural killer T cells, neutrophils, and plasmacytoid dendritic cells were all linked to COVID-19 and PH. The highest Pearson correlation was reported between CCL20 and plasmacytoid dendritic cells, and the highest connection between SELE and activated dendritic cells. The immunological response of different cells may be regulated by SELE and CCL20, leading to an increase in the incidence of COVID-19 coupled PH. Lymphocytes, including NK cells, are activated and migrate to the lung during an acute coronavirus infection because of the accumulation of inflammatory mononuclear macrophages and neutrophils, which release cytokines and chemokines. When SELE and CCL20 levels drop, NK cell numbers and function decline. PH developed as a result of an increase in sensitivity to COVID-19 and modification of pulmonary artery walls caused by NK cell destruction. Chronic tissue inflammation can be caused by CD4 + T cell-mediated cellular immunity, which is a particular cellular immune response driven by CD4 + T cells47,48,49,50. There is an invasion of lymphocytes (mostly T cells) and mononuclear phagocytic cell lines, resulting in an exudative inflammation. The pathophysiology of COVID-19-complicated PH is also influenced by SELE and CCL20-mediated neutrophil proliferation. By secreting neutrophils extracellular traps, which in turn increase pulmonary artery endothelial cell damage and smooth muscle cell proliferation, neutrophils serve to both perpetuate and worsen inflammation.

SELE, CCL20 with dendritic cells exhibited the greatest correlation among the five cell types, according to a Pearson analysis. Dendritic cells were dramatically decreased in individuals with COVID-19 sequelae, according to research by Tomonari Sumi et al.51. About 7 months after SARS-CoV-2 infection, Perez-Gomez A et al52 discovered that dendritic cells dropped considerably in vivo. There are parallels between this conclusion and our own. There is a decrease in the number of mature myeloid DC and associated functional abnormalities in people with COVID-19 who also have PH. Therefore, DC cells are unable to initiate an immune response by encouraging primary T cell activation and proliferation in order to protect the body from virus-induced harm.

In addition to diagnosis, the identification of drugs targeting pathogenesis is also an important direction for us to explore. Current treatment of COVID-19 is primarily dependent on supportive care, together with the use of antiviral and immunomodulatory drugs. Given the distribution of the population living with comorbidities, specifically, the predominantly middle-aged and elderly demographic, poly pharmaceuticals and drug-drug interactions might be apparent. Unfortunately, the potential risk of drug-drug interactions is largely unknown since most studies on COVID-19 do not provide details on interactions between drugs used in the course of COVID-19 treatment and co-medications used for the management of other comorbidities in these patients53. Moreover, the use of some commonly used drugs in COVID patients may lead to an increased number of adverse pulmonary effects54. The current direction is to find suitable drugs that have a certain therapeutic effect and relatively few side effects. In this study, because of the significance of SELE and CCL20 in the pathogenesis of COVID-19 and PH, we chose them as drug prediction and molecular docking targets. The binding energy of these two molecules with FENRETINIDE, 1-NITROPYRENE, and AFLATOXIN B1 was the lowest of any of the eight anticipated medications, which was not expected. At present, there are few reports that 1-NITROPYRENE and AFLATOXIN B1 can improve the symptoms of COVID-19 and PH55,56,57,58,59,60,61,62.Also as a nitro compound, WenXia Feng et al. found that Inhaled nitric oxide treatment was beneficial in reducing and stabilizing the PASP and might also reduce the risk of right heart failure in COVID‐19 with pulmonary hypertension63. In this study, we found that whether in the enrichr database or the CTD database, by performing drug predictions on the key genes that COVID combines with PH, we found that we could predict that 1-NITROPYRENE was a potential therapeutic drug, and that through molecular docking and molecular dynamics simulations, we found that the combination of 1-NITROPYRENE and the two key genes was extremely stable. Therefore, it has great potential to be used as a drug to treat this complication. Because of its beneficial effects on glucose tolerance, lipid levels, and body fat percentage, the synthetic retinide derivative Fenretinide has been used for a variety of medical purposes, including cancer prevention and treatment, atherosclerosis improvement, and the amelioration of non-alcoholic fatty liver disease. Its capacity to reduce the production of inflammatory mediators and prevent macrophage polarization may be the primary mechanism at work here63,64,65. Fenretinide was found to inhibit the release of pro-inflammatory factors (IL-1β, MCP1, iNOS, and TNF-α). Fenretinide may inhibit NF-κB signaling by reducing the nuclear translocation of the protein via downregulation of IKKβ and IκBα phosphorylation64. In addition, delayed release of IFN-I is well known in SARS-CoV infection as a mechanism that slows the antiviral response of the body. The viral mechanisms associated with IFN-I evasion are multifaceted, including sequestering and shielding RNA within double-membrane vesicles, modification of viral mRNA 5-cap structures, and specific targeting of antiviral cellular pathways. In SARS-CoV and MERS-CoV, IFN-I production is protective only at the early stages after infection; at later time points, on the contrary, when the immune response is increased, IFN-I and inflammatory cytokines become pathogenic66,67,68,69. In both zika virus and dengue virus, fenretinide inhibited the non-structural protein 5 (NS5), which contributed to virulence, by preventing the production of IFN-I70. Thus, it is possible that fenretinide may also influence the mechanisms regulating IFN-I evasion in coronavirus infections. However, there are still few relevant findings, and the precise mechanism of action is unclear, calling for more in-depth pharmacological study.

Our literature search found a lack of research on the shared mechanism between COVID-19 and PH, especially bioinformatic studies. Here, we screened for C-DEGs, tested Core genes with a machine algorithm, and built a model to predict the COVID-19 combined with PH. We investigated how these two essential genes are linked to diseases and made predictions about the transcription factors and miRNAs that regulate them. In the end, we used molecular docking and targeting predictions for two important genes to determine which medications would be most effective.

However, there were certain gaps in our research. First, through molecular docking, we found that Fenretinide, a targeted drug for SELE and CCL20, may be a new target for the treatment of COVID 19 combined PH, though the mechanism by which it acts still needs to be further studied. External validation is needed for additional verification of the current outcomes. Moreover, in vitro model validation is required to additionally explore the core gene functions.