Discussion
VPA is a well-known HDAC2 inhibitor, which has been safely used for over 50 years as an anti-convulsant drug.[8-10] VPA is taken-up by endothelium immediately within a minute of intravenous injection in humans.[23] Cardiovascular complications with wide-spread endothelial dysfunction, thrombosis and endothelitis are rapidly emerging as a key threat in COVID-19 in addition to respiratory disease.[1, 2] Endothelium plays a central role in inflammation, thrombosis and cardiovascular complications, and a SARS-CoV-2 use ACE-2 receptors for cell entry, which is mainly expressed on ECs.[2, 11, 12] Altogether, these information indicate for a bigger role for VPA and endothelium in COVID-19. Accordingly, to devise therapeutic strategies to counteract SARS-CoV-2 infection and the associated COVID-19 pathology, it is crucial to understand, and to apply the presented and other published knowledge towards repurposing already approved drugs to treat or prevent COVID-19. Accordingly, we plan to evaluate the role of potential drug VPA on ACE-2 expression in ECs. We also discuss our finding in relation to other published article providing a plausible mechanism for the use of VPA for prevention and treatment of COVID-19 (Fig. 2 ).
Our data demonstrate that VPA treatment to ECs significantly reduced ACE-2 expression. This data is clinically very relevant for COVID-19 as ACE-2 is a cell “entry door” for SARS-Co-2 and also because SARS-CoV-2 infection is shown to be enhanced by over-expression[24] and diminished by inhibition of ACE-2.[25-27] The effect of VPA on ACE-2 expression indicates that VPA can inhibit the SARS-CoV-2 rate of infection by reducing its receptor ACE-2 expression level and can be used as a prevention strategy against COVID-19. Next, our data on reduced expression of IL-6 in VPA-treated ECs is also clinically relevant, as the cause of death is the inflammation and thrombosis due to the “cytokine storm” mainly of interleukins such as IL-6 in the lungs of COVID-19 patients.[3] Particularly, level of IL-6 predicts respiratory failure and IL-6 inhibitors are proposed to ameliorate severe lung damage in COVID-19 patients.[28] ECs can secrete pro-inflammatory cytokines and these cytokines “activate” endothelial cells to produce tissue factor, which regulates thrombosis.[29, 30] ICAM-1 is markers for endothelial “activation”.[30] Interestingly, VPA also significantly reduced ICAM-1 expression indicating reduced endothelial activation (Fig. 1E ). The presented data is of immense importance as it provides a “treatment” strategy for COVID-19 as VPA-induced down-regulation of IL-6 and ICAM-1 are the regulatory molecules implicated in SARS-CoV-2-induced inflammation and coagulation.
PCR array analysis for human endothelial-related genes in VPA-treated ECs is reported with a total 14 significantly up- and 14 down-regulated genes.[19] To evaluate the therapeutic role of VPA in COVID-19, it is important to look at these VPA-induced differentially expressed endothelial genes in relation to inflammation, coagulation, endothelial function, endothelitis, cardiovascular and respiratory diseases. The most up-regulated endothelial genes which are relevant to COVID-19 prevention and/or treatment include BNP (Natriuretic peptide B; 145-fold), MMP-9 (Matrix metallopeptidase 9, 121-fold), PF-4 (Platelet factor 4, 25-fold), T-PA (Plasminogen activator, tissue, 18-fold), COX-2 (Prostaglandin-endoperoxide synthase 2, 3-fold) and TGFβ1 (Transforming growth factor, beta 1, 1.3-fold). BNP was the most up-regulated gene in VPA-treated ECs, which is a diagnostic biomarker for cardiac dysfunction,[31] but is also anti-inflammatory and inhibit interleukins.[32, 33] The next up-regulated gene MMP-9 is anti-inflammatory[34, 35] and is associated with lung remodelling.[36] PF-4 is anti-viral as it inhibits human immunodeficiency virus type 1 (HIV-1) infection,[37] and also protects lung against bacterial infection.[38] VPA-induced endothelial and anti-thrombotic T-PA is of particularly relevance to the COVID-19 patients. T-PA treatment is suggested for COVID-19 Associated Acute Respiratory Distress Syndrome (ARDS).[39] Furthermore, VPA is known to have protective effects in severe hemorrhage and ischemia-reperfusion injury.[40] Coagulopathy has become a hallmark of severe COVID-19 with high rates of central line thrombosis and vascular occlusive events (e.g. ischemic limbs, strokes, etc).[39] Ventilator-induced lung injury is another cause in COVID-19 death and T-PA is known to attenuate ventilator-induced lung injury.[41] Overall, VPA-induced T-PA appears to be beneficial to the COVID-19 patients due to its anti-thrombotic activity and attenuation of ventilator-induced lung injury function. COX-2 was also up-regulated in VPA-treated ECs. Given the important role played by COX-2 in antipyretic nonsteroidal anti-inflammatory drugs (NSAIDs), caution should be taken while prescribing NSAIDs with VPA to COVID-19 patients.[42] The most down-regulated and relevant to COVID-19 prevention and/or treatment genes include TFPI (Tissue factor pathway inhibitor, 6-fold) and SPHK (Sphingosine kinase 1, 5-fold). Tissue factor as an initiator of coagulation and inflammation in the lung.[43] Most down-regulated anti-coagulant TFPI activities are reported in the lungs of idiopathic pulmonary fibrosis patients,[44] and it is marker for the prediction of deep venous thrombosis and tumor metastasis in patients with lung cancer.[45] Given the increased rate of thrombosis in COVID-19, the status of TFPI needs to be investigated with VPA-treatment. The VPA-induced down-regulation of SPHK1 appears to help COVID-19 treatment as SPHK1 is known to contributes to ventilator-associated lung injury,[46] elevated SPHK1 enhances influenza virus infection,[47] and SPHK1 also serves as a pro-viral factor by regulating viral RNA synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection.[48] VPA-induced down-regulation of SPHK appears to help COVID-19 patients.
The exact mechanism behind HCQ-mediated therapeutic benefit to COVID-19 patients is not completely elucidated but what we know as a fact that HCQ is an autophagy inhibitor.[17] TGFβ-signalling induces EndMT,[16] and loss of endothelial autophagy[18] or VPA-treatment to ECs,[19] both exhibit TGFβ-induced EndMT. It is quite possible that EndMT-associated loss of endothelial function and gain of pro-fibrogenic mesenchymal function is the mechanism behind HCQ-associated benefit in COVID-19 patients. In that scenario, similar benefit can be expected from VPA in COVID-19 patients. EndMT is known to play roles in the development process,[49] and also in diseases such as pulmonary vein stenosis, anomalous vascular remodeling, cerebral cavernous malformations, cancer progression and organ fibrosis.[50, 51] Most importantly, It was reported that 16% of the lung fibroblasts in pulmonary fibrosis mouse model originated from ECs through EndMT process,[52] and loss of endothelial autophagy-induced EndMT exacerbated lung fibrosis in the same mouse model.[18] Role of EndMT in exacerbation of lung fibrosis might be the reason behind no-effect or adverse effect of HCQ in critically-ill COVID-19 patients, however this needs to be evaluated. Similar, side effect can be expected for VPA, and if true, them precautions should be taken before prescribing VPA to COVID-19 patients with pre-existing pathologies. Furthermore, VPA is an anti-seizure drug and there are reports suggesting that patients with pre-existing seizure disorder may be at greater risk for getting breakthrough seizures.[53] In these COVID-19 patients, VPA can be a preferred treatment option.
This new knowledge about “mechanism of action” of VPA provides a novel potential therapeutic drug target for prevention and treatment of COVID-19, and warrants immediate further investigation in animal models and in humans. We also suggest that VPA based therapeutics may exacerbate EndMT-related pathologies in COVID-19 patients with pre-existing diseases, and promote personalized therapy in such COVID-19 patients. Overall, our manuscript provides a better source of necessary knowledge on VPA for reasearchers and health care practitioners to find treatment for COVID-19. VPA is a FDA approved drugs and any proof of these drugs showing protective effect against COVID-19 will reduce the required drug approval time and it can be fast-tracked for testing against COVID-19.