Neurological consequences of COVID-19 still pose a relevant puzzle to medical and scientific community. Since its first cases, CNS invasion has been described (Paterson et al., 2020; Sanchez et al., 2021) but the routes and mechanisms by which SARS-CoV-2 gains entry to brain parenchyma remain allusive (Brann et al., 2020). In the present study we have investigated the molecular indices of Neuro-COVID-19 as they are related to brain endothelial cells forming the BBB. Despite our previous observation that primary HBMECs express several receptors for the virus (Torices et al., 2021), we found little to no indication of productive viral replication in the supernatants of HBMECs which is in accordance with previous reports that described that several endothelial cell types are not permissive for SARS-CoV-2 productive infection (McCracken et al., 2021; Nascimento Conde et al., 2021). Krasemann et al., (2022) observed infection of iPS-derived HBMECs but only at MOIs 10 and 100, which are unlikely to have pathological significance.
Exposure to SARS-CoV-2 led to augmentation of ACE2 and TMPRSS2 expression, which is consistent with our previous observations, in which HBMECs were exposed to the S1 subunit of Spike protein (Torices et al., 2021). Despite the apparent lack of productive infection, SARS-CoV-2 induced apoptotic death of HBMECs in similar levels as compared to Vero epithelial cells and to elsewhere described in the literature (Heuberger et al., 2021). In fact, HBMECs have been shown to undergo apoptotic cell death in response to viral infections, including Dengue (Meuren et al., 2021) and Zika (Leda et al., 2019; Mladinich et al., 2021) viruses, followed or not by changes in BBB permeability. These results suggest that interaction of host cells with viral surface proteins may be sufficient to trigger cellular apoptosis induction even in the absence of productive infection.
Tight junction proteins have a crucial role on maintaining BBB integrity and its selective paracellular permeability (reviewed by Takata et al., 2021). In our study, SARS-CoV-2-infected Vero E6 cells showed a marked disorganization of paracellular tight junctions, as shown by ZO-1 immunostaining. Previous studies described that treatment of HBMECs in 2D or 3D cultures with the S1 subunit Spike1 protein led to mislocalization of ZO-1, concomitantly with cytokine secretion (Buzhdygan et al., 2020; DeOre et al., 2021). Not only ZO-1 (Hao et al., 2020), but also β-catenin, cadherin-5 and occludin junctional proteins have recently been shown to be affected by SARS-CoV-2 proteins in HUVECs (Rauti et al., 2021). ZO-1 possesses a PDZ domain, which is responsible for binding and interaction with other proteins (Saras & Heldin, 1996; Giepmans & Moolenaar, 1998). Interestingly, ACE2 possesses a PDZ-binding domain (Dasgupta & Bandyopadhyay, 2021; Caillet-Saguy & Wolff, 2021), and it has been suggested that epitopes of viral proteins, such as 1–60: M1Lys60 and 241–300: Ala240-Glu300 could directly bind to ZO-1 and VCAM-1 PDZ domains, thus suggesting a possible alternative route of CNS entry. We found ZO-1 and claudin-5 protein levels to be increased after 24 h of infection in HBMECs and this increase is consistent with what our group recently demonstrated with S1 treatment (Torices et al., 2021). Whether ZO-1 increase can be directly related to BBB permeability may vary among experimental models and infectious agents. We have described that the Honduras isolate of Zika virus selectively up-regulates ZO-1 expression in vitro, while BBB permeability was increased in vivo (Leda et al., 2019). In fact, proper BBB functioning relies on the combined expression and localization of ZO-1, -2 and − 3, claudin-5 and occludin (Feng et al., 2018). Direct infection with higher MOIs of SARS-CoV-2 or treatment with plasma from COVID-19 patients failed to induce significant increases in permeability in BMECs in vitro (Constant et al., 2021). Conversely, using the K18 mouse model and hamster infection, Zhang et al. (2021) showed that SARS-CoV-2 effectively infects and replicates in HBMECs, but leads to no change in BBB permeability and TJ proteins. Interestingly, a massive inoculation of iPS-derived HBMECs (MOIs 10 and 100), showed active viral replication, whereas MOIs 0.1 and 1 described infectivity near to 0.6% cells, which is similar to what we observed herein (Krasemann et al., 2022).
Following the initial characterization of infectivity profile in HBMECs, we sought to characterize the transcriptomic landscape of SARS-CoV-2 infection in BBB-forming cells. We performed RNA-Seq analyses of SARS-CoV-2-infected cultures at 6 and 24 hpi, with MOIs 0.1 and 0.01. Due to the minimal change in the overall host cell transcriptome with most of the experimental conditions used in this study, we focused our subsequent analyses to the MOI 0.1 infection at 24 hpi. The majority of the significantly up-regulated genes corresponded to known endothelial activation pathways, including CXCL1, -2, -3 and CCL20, PTX3, ICAM1 and TNF. Interestingly, the overexpression of the LTB-TNF-RELB ensemble by SARS-CoV-2, provided evidence that activation of the non-canonical NF-κB pathway activation may be taking place. NF-κB is a family of transcription factors that can be activated by several ligands and activates the expression of proinflammatory cytokines and chemokines (Sun et al., 2012). Interestingly, the main protease of SARS-CoV-2 (Mpro) cleaves a member of the NF-κB family, NEMO, which in turn leads to HBMEC cell death in vitro and in vivo (Wenzel et al., 2021). RELB can form heterodimers with p50/p105, p52/p100 and p65 (Shih et al., 2012; reviewed by Mockenhaupt et al., 2021), but can also bind to sirtuin1 to direct epigenetic silencing of inflammatory gene expression (Chen et al., 2009; Liu et al., 2011). In fact, among KEGG pathways enriched in our datasets, we found ribosomal structure and function as possible candidates for epigenetic regulation induced by SARS-CoV-2. These observations suggest that host epigenetic factors may be key for the outcome of COVID-19 and/or long COVID-19. Indeed, the promoters of the genes involved in inflammation, including NF-κB, can be demethylated, thereby resulting in an increased expression of interferons (IFNs), possibly leading to “cytokine storm” (Coit et al., 2016). The expression of IL-6, another important player in the so-called cytokine storm occurring in the most severe COVID-19 patients, was also significantly increased in infected HBMEC and it is known to be modulated by methylation of its promoter. It was also observed that oxidative stress induced by viral infections, including SARS-CoV-2 infection, can inhibit the maintenance of DNA methyltransferase DNMT1, thereby aggravating the DNA methylation defects (Li et al., 2014; Perl, 2013; Sawalha et al., 2020). Our preliminary results (not shown) indicate that SARS-CoV-2 infection of HBMEC results in a decrease in DNA methylation, supporting a recent study of genome-wide DNA methylation analysis in peripheral blood of COVID-19 infected individuals, which identified marked epigenetic signatures, such as hypermethylation of IFN-related genes and hypomethylation of inflammatory genes (Corely et al., 2020). Such observations further suggest the involvement of epigenetic regulatory mechanisms in COVID-19 (Mantovani A & Netea, 2020).
As stated above, PTX3 was one of the main hits found in the transcriptomic analyses. Pentraxins are a superfamily of multifunctional proteins with conserved phylogeny (Daigo et al., 2014), divided into 2 groups based on their primary structure: short and long pentraxin, where c-reactive protein and PTX are examples of short and long pentraxins, respectively. PTX3 is expressed in several neural cell types (Muffley et al., 2012; Shindo et al., 2016; Freezer et al., 2017; Siqueira et al., 2018; Wesley et al., 2022) and in endothelial cells can be upregulated by inflammatory stress such as cytokine stimulation (Breviario et al., 1992). We performed a profiling of angiogenesis-related panel in the supernatants of SARS-CoV-2-infected HBMEC and confirmed that PTX3 was increased by infection. PTX3 is known to be produced in high amounts by blood vessels in vascular inflammatory conditions (Fazzini et al., 2001) and inhibits FGF2-dependent angiogenesis (Rusnati et al., 2004; Presta et al., 2018). Pathological vascularization and angiogenesis have been described as a unique comorbidity associated with SARS-CoV-2 infection in the pulmonary endothelium (Meini et al., 2020), including microvascular distortion and increased intussusceptive angiogenesis (Ackerman et al., 2020; Mentzer et al., 2022). Moreover, VEGF as well as other angiogenic-related analytes were found to be increased in COVID-19 patients (including PTX3), which correlated with disease severity (Maldonado et al., 2022). Accordingly, VEGF levels were 8% increased in the supernatants of infected HBMECs, even though vegf transcripts remained unaltered. It is well-known that inflammation, especially IL-6-dependent, can stimulate defective angiogenesis (Gopinathan et al., 2015) and our data further contributes to the notion that following SARS-CoV-2 infection there is an intense brain endothelial activation which leads to defective angiogenic signaling and possibly endothelial permeability. Additionally, we found HIF-1α to be greatly increased after 24 hpi. HIF-1α is a major angiogenesis inductor and is known to be up-regulated by distinct viral infections (reviewed by Reyes et al., 2020). HIF-1α is activated and translocated to the nucleus upon hypoxic conditions (Ke & Costa, 2006) and it has been shown that COVID-19 patients present massive hypoxia due to vasoconstriction and coagulopathy (Afsar et al., 2020). Interestingly, ACE2 expression is decreased in pulmonary smooth muscle cells upon HIF-1α accumulation (Zhang et al., 2020), whereas hypoxia leads to a biphasic modulation of both ACE2 and TMPRSS2 expression on brain microvascular endothelial cells (hCMEC/D3), with an initial increase at 6 h and a decrease at 48 h of hypoxic stimulus cells (Imperio et al., 2021). These observations are in accordance with our present data, that ACE2 is decreased while HIF-1α is increased at 24 hpi. Although VEGF is one of the most described downstream targets of HIF-1α activation, apoptotic cell death and IFN-stimulated gene expression are additional targets of HIF-1α activation (Reyes et al., 2020), which can also be dependent on NF-κB signaling pathway (Walmsley et al., 2005). Our data indicates that HIF-1α up-regulation can be a part of a SARS-CoV-2-induced endothelial activation, along with cytokine/chemokine stimulation and NF-κB non-canonical activation.
Our final series of experiments focused on mitochondrial morphology and dynamics in HBMECs following SARS-CoV-2 infection. It is well known that mitochondria are gatekeepers of BBB endothelium physiology and correspond to higher cytoplasmic volume as compared to non-cerebrovascular endothelial cells (Oldendorf et al., 1977; reviewed by Parodi-Rullan et al., 2021). Moreover, mitochondrial function is important for BBB maintenance and integrity (Doll et al., 2015). We first employed a morphological/morphometrical approach to determine the mitochondrial contents and cellular distribution. Herein we demonstrated that direct exposure to SARS-CoV-2 led to a remodeling of mitochondrial networks. By using the MiNA plugin, we verified that infected HBMECs had increased mitochondrial footprint, as an estimation of overall TOMM20 pixel signal. Recent reports have also shown an effect of SARS-CoV-2 and COVID-19 on mitochondrial biology: monocytes isolated from COVID-19 patients display reduced mitochondrial membrane potential and SARS-CoV-2 viral load was positively correlated with generation of ROS (Romão et al., 2022). Importantly, endothelial cells exposed to SARS-CoV-2 Spike1 protein showed decreased tubular and increased fragmented mitochondrial networks in vitro, which was accompanied by a decrease in oxygen consumption rate and increase extracellular acidification rate (Lei et al., 2021). Confirming observation was recently described by Domizio et al. (2022), in which pulmonary endothelial cells in a lung-on-a-chip infection model displayed increased mitochondrial networks. Similarly, we demonstrated that mitochondrial networks were increased, as determined by summed branch length analyses, which indicates that SARS-CoV-2-infected HBMECs had longer mitochondrial ramifications. Changes in endothelial mito-morphology are well described in several models of inflammatory diseases and/or aging (Burns et al., 1979; Jendrach et al., 2005; Forrester et al., 2020) and are correlated with abnormalities in mitochondrial quality control system, which in turn can lead to increased ROS production. Mitochondrial quality control encompasses biogenesis, fission, fusion and mitophagy processes, which are essential for its biology and function. We analyzed markers of fusion and fission processes in SARS-CoV-2-infected cultures and determined that while MOI 0.01 led to an increase in fission-related gene expression (Fis1 and Drp1), this effect was not observed in protein levels or phosphorylation. However, mitofusin2 protein contents were found to be increased in MOI 0.1-infected cultures, which could explain the increased values in branch lengths. Moreover, we found mitochondria associated to some extent to multivesicular bodies, which has also been described as another pathway for mitochondrial quality control (Sugiura et al., 2014; Picca et al., 2020). Interestingly, several reports have linked NF-κB mediated inflammation with mitochondrial responses (Nakahira et al., 2011; Zhou et al., 2016; Zhong et al., 2016; Liu et al., 2018), which could indicate that in fact mitochondrial remodeling observed in infected HBMECs could be due to (or lead to) SARS-CoV-2-induced inflammatory response.