Abstract
We hypothesized that exosomal microRNAs could be implied in the pathogenesis of thromboembolic complications in coronavirus disease 2019 (COVID-19). We isolated circulating exosomes from patients with COVID-19, and then we divided our population in two arms based on the D-dimer level on hospital admission. We observed that exosomal miR-145 and miR-885 significantly correlate with D-dimer levels. Moreover, we demonstrate that human endothelial cells express the main cofactors needed for the internalization of the "Severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), including angiotensin converting enzyme 2, transmembrane protease serine 2, and CD-147. Interestingly, human endothelial cells treated with serum from COVID-19 patients release significantly less miR-145 and miR-885, exhibit increased apoptosis, and display significantly impaired angiogenetic properties compared with cells treated with non-COVID-19 serum. Taken together, our data indicate that exosomal miR-145 and miR-885 are essential in modulating thromboembolic events in COVID-19.
SIGNIFICANCE STATEMENT This work demonstrates for the first time that two specific microRNAs (namely miR-145 and miR-885) contained in circulating exosomes are functionally involved in thromboembolic events in COVID-19. These findings are especially relevant to the general audience when considering the emerging prominence of post-acute sequelae of COVID-19 systemic manifestations known as Long COVID.
Introduction
COVID-19 has caused an enormous number of deaths due to the poor information on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its exact mechanisms of action. Substantial progresses have been recently made in science and technology, improving the management of patients with COVID-19 (Kaur and Gupta, 2020; Stasi et al., 2020; Tregoning et al., 2020; Ulinici et al., 2021; Gupta et al., 2022; Närhi et al., 2022; Vincent et al., 2022; Weerakkody et al., 2022). However, the war is not over yet, inasmuch as the COVID-19 pandemic leaves substantial aftermaths due to the long-term complications of the disease, which are often disabling, reducing the quality of life (Crook et al., 2021; Iqbal et al., 2021; Michelen et al., 2021; Desai et al., 2022; Robineau et al., 2022; Whitaker et al., 2022). Several reports suggest that the risk of death for COVID-19 survivors is higher than the risk associated with other conditions, due at least in part to long-term complications (Davido et al., 2020; Basu et al., 2021; Moreno-Pérez et al., 2021; Yang et al., 2021; Zhang et al., 2021b; Comelli et al., 2022; Smith, 2022). Of note, the number of deaths for long-term COVID-19 complications has not necessarily been recorded as deaths due to COVID-19; therefore, the actual situation could be worse than what was reported. In this context, the identification of useful biomarkers of fatal complications is sorely needed.
The symptoms of patients with COVID-19 vary greatly, ranging from an asymptomatic state to debilitating respiratory failure due to bilateral pneumonia (Çalica et al., 2020; Iacobucci, 2022; Kaliszewski et al., 2022). Patients can also develop a systemic inflammatory state that favors multiorgan failure and increases susceptibility to systemic thromboembolic complications that may contribute to a rapid clinical deterioration (Mui et al., 2021). Arterial thrombotic events include end-organ ischemia to systemic organs, cerebrovascular accidents, and limb ischemia, and are associated with high D-dimer, prolonged prothrombin time (PT), and elevated levels of fibrinogen, which indicate activation of coagulation pathways and thrombosis (Li et al., 2020; Gambardella et al., 2021). D-dimer, in particular, is one of the most sensitive coagulation parameters in COVID-19 and indicates a greater risk for the development of thrombosis (Zhang et al., 2020; Conte et al., 2021; Ozen et al., 2021; Poudel et al., 2021). Thus, D-dimer measurement is currently considered a critical approach in the clinical management of COVID-19 (Rostami and Mansouritorghabeh, 2020; Conte et al., 2021; Ghosh and Ghosh, 2022).
A finding that emerged from the intensive research on COVID-19 is that the endothelium is a key target organ of COVID-19. We were among the first groups to describe the involvement of endothelial dysfunction in COVID-19 (Sardu et al., 2020), and successively both clinical and preclinical evidence supported our finding (Otifi and Adiga, 2022). The endothelium is instrumental in thrombosis, fibrinolysis, inflammation, and in maintaining a proper vasodilation/vasoconstriction and antioxidant/pro-oxidant balance (Wu and Thiagarajan, 1996; Libby et al., 2006; Gambardella et al., 2020; Adebayo et al., 2021); therefore, an impaired endothelial function could be the mechanism underlying the systemic complications of COVID-19, especially but not exclusively thromboembolic events (Sardu et al., 2020; Adebayo et al., 2021).
Direct effects of COVID-19 as well as indirect effects of the infection (inflammation, hypoxia) might predispose patients to thrombotic events (Bikdeli et al., 2020) that then play a decisive role in the clinical outcome (Cryer et al., 2022). Thus, an early biomarker of the development of thromboembolic events and predictor of associated clinical outcomes could be useful for a timely intervention with targeted therapies.
In this context, measuring the levels of microRNAs (miRNAs), freely circulating or within extracellular vesicles, represents a useful strategy as diagnostic and prognostic biomarker in numerous disease states (Cho et al., 2021; Guo et al., 2021; He et al., 2021; Ning et al., 2021; Ueta et al., 2021; Ying et al., 2021; Zhang et al., 2021a; Lin et al., 2022; Lyu et al., 2022; Mahmoudi et al., 2022; Wang et al., 2022). The microRNA cargo of extracellular vesicles could not only contribute to the pathogenesis of thrombotic and thromboembolic complications of COVID-19 but most likely also be a diagnostic/prognostic marker. We recently identified a significant association linking endothelial exosomal miR-24 and cerebrovascular disorders, indicating that this approach could be an extremely useful tool for diagnosis and prognosis (Gambardella et al., 2021). Herein, we aim at identifying specific circulating miRNAs associated with thromboembolic events in patients with COVID-19.
Materials and Methods
To test our hypothesis that exosomal miRNAs are a major determinant of thrombosis in COVID-19, we enrolled 26 patients positive for COVID-19 admitted to the Sant’Anna and San Sebastiano Hospital of Caserta and Naples University (Italy). The serum from 10 non-COVID-19 subjects was used as control. All subjects underwent a SARS-CoV-2 test by reverse-transcription polymerase chain reaction to rule out or confirm the COVID-19 diagnosis. The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments; we obtained a written informed consent from all participants or their legal representatives.
We isolated circulating exosomes from equal amounts of serum, as previously reported by our research group (Gambardella et al., 2021). Purity and absence of contamination were assessed by immunoblot (Wang et al., 2020b), whereas morphology and size distribution were examined by dynamic light scattering and electron microscopy (not shown). Levels of miRNAs were quantified by reverse-transcription polymerase chain reaction (Morelli et al., 2019; Wang et al., 2020b; Gambardella et al., 2021).
In Vitro Experiments
We performed in vitro assays in human umbilical vein endothelial cells (HUVECs), cultured in F12 medium enriched with specific growth factors for endothelial growth (Lonza). The cells were cultured at 37°C in 95% air and 5% CO2. All experiments were performed at least in triplicate using cells between passages 5 and 9. The experimental protocol on HUVECs consisted of 24 h incubation with serum from patients with COVID-19 or from patients negative for SARS-COV-2 as control. The serum was used with a final dilution of 1:50 directly in the medium.
Angiogenesis Assay
The formation of network-like structures by HUVECs on extracellular matrix-like 3D gel was performed as previously described (Santulli et al., 2011; Gambardella et al., 2018). HUVECs (5 × 104) were seeded on Matrigel Matrix and incubated at 37°C for 24 h, in presence of COVID-19 serum or control serum.
Lipid Peroxidation Assay
The level of malondialdehyde was measured by using a lipid peroxidation assay Kit (#ab118970, Abcam, Cambridge, UK), as we previously described (Tang et al., 2021).
Immunoblot Analysis
Total lysates were prepared as we described (Sorriento et al., 2009). Immunoblot analyses were performed as previously reported (Wang et al., 2020b). Briefly, lysates were electrophoresed by SDS-PAGE and transferred to nitrocellulose. Angiotensin converting enzyme 2, transmembrane protease serine 2, CD-147, cleaved caspase 3, and glyceraldehyde-3-phosphate dehydrogenase were visualized by specific antibodies (Cell Signaling Technology, Danvers, MA), whereas fluorochrome-conjugated anti-rabbit and anti-mouse were used as secondary antibodies (LI-COR, Lincoln, NE). The nitrocellulose membrane with fluorescent signal was scanned using the LI-COR imaging system, as we described (Matarese et al., 2020a; Dridi et al., 2022).
Statistical Analysis
Data are expressed as mean ± S.E. All data were analyzed using GraphPad Prism version 9 (GraphPad by Dotmatics, Boston, MA) with a significant difference established at a P value < 0.05. The normal distribution of values was verified by the Shapiro-Wilk test; the Student’s 2-tailed t test was applied to compare values between groups. The correlation between miRNA and D-dimer levels was determined by Pearson's correlation analysis.
Results
miR-145 and miR-885 Downregulation was Associated with Thrombotic Risk and Mortality in Patients with COVID-19
To verify our hypothesis that exosomal miRNAs play a crucial role in the pathogenesis of thrombosis in COVID-19, we divided our population in two groups based on the serum D-dimer level on hospital admission, using a cut-off of 3 μg/ml. We did not detect any significant difference in the main clinical characteristics when comparing patients with low versus high D-dimer. Strikingly, we found that exosomal miR-145 and miR-885 were significantly downregulated in the subjects in the high D-dimer arm compared with subjects in the low D-dimer arm (Fig. 1, A and B).
miR-145 and miR-885 are associated with a higher risk of thrombosis and mortality in patients with COVID-19. Expression level of exosomal miRNAs in patients with COVID-19. Different levels of miRNAs (miR-145, miR-885) in patients with high D-dimer and low D-dimer (A–B); in the violin plots, median (solid line) and quartiles (dotted lines) are indicated. The trend was confirmed by linear regression analysis (C–D). In panels E and F, miRNAs levels are quantified as mean ± S.E. among survivors and non survivors (E–F); *P < 0.001.
The Pearson’s correlation analysis confirmed these findings (Fig. 1, C and D). Furthermore, when we dichotomized our COVID-19 population in survivors and non-survivors, we noted that the levels of both miR-145 and miR-885 were significantly lower in the patients who did not survive (Fig. 1, E and F). These data indicate that the downregulation of miR-145 and miR-885 is associated to worst prognosis, correlating with a higher thrombotic risk and mortality in COVID-19. It is noteworthy that miRNAs were assessed on hospital admission; hence, their alterations already at time 0 denote miR-145 and miR-885 as powerful prognostic predictors of adverse outcome.
Endothelial Dysfunction as Responsible for miR-145 and miR-885 Downregulation
Endothelial cells are leading actors in producing and releasing tissue factor (TF) and von Willebrand Factor (vWF), thus regulating the thrombotic cascade. We hypothesized that the endothelial dysfunction occurring during SARS-CoV-2 infection determines a dysregulation of this mechanism, reducing the capability of endothelial cells to release miR-145 and miR-885, eventually resulting in an uncontrolled coagulation. To test this hypothesis we employed human endothelial cells as an in vitro model and exposed them to serum from patients with COVID-19, compared with cells exposed to serum from patients negative for SARS-CoV-2.
Importantly, HUVECs expressed all the major SARS-CoV-2 receptors, including angiotensin converting enzyme 2, transmembrane protease serine 2, and CD-147 (Matarese et al., 2020b; Wang et al., 2020a; Zipeto et al., 2020; Evans and Liu, 2021), providing further evidence of the susceptibility of endothelial cells to SARS-CoV-2 infection (Fig. 2A). This expression pattern of viral receptors also confirms that our in vitro setting is a valuable model to study endothelial response and damage in COVID-19.
Endothelial cell damage induced by COVID-19 environment. Representative immunoblots showing the expression of the main viral receptors on human endothelial cells (A). The exposure of HUVECs to COVID-19 serum induces the cleavage of caspase 3, indicating apoptosis activation (B), quantified in panels C and D. Data are from triplicate experiments; *P < 0.001.
The exposure to COVID-19 serum induces apoptosis of endothelial cells, as indicated by caspase 3 activation (Fig. 2, B–D), supporting the hypothesis of a significant endothelial damage.
We further explored the endothelial dysfunction evaluating a key feature of viable endothelial cells, angiogenic competence. Consistently, we observed an impaired capacity of forming network-like structures on Matrigel, indicating that COVID-19 serum exposure compromises the angiogenic capacity of endothelial cells (Fig. 3, A and B).
Endothelial cell angiogenic capacity is impaired in COVID-19. Angiogenesis assay on Matrigel. COVID-19 serum significantly affects the angiogenic capacity of endothelial cells. Representative pictures of network-like formation (dimensional bar: 100 μm) from independent triplicate experiments (A) and relative quantification (B); *P < 0.001.
To assess the effects of COVID-19 on lipid peroxidation, an established hallmark of severity in patients with COVID-19 (Martín-Fernández et al., 2021; Žarković et al., 2021; Lage et al., 2022; Soto et al., 2022), we measured malondialdehyde level in HUVEC lysate, and we observed a significantly increased peroxidation in the lysate of endothelial cells incubated for 24 hours with COVID-19 serum compared to cells incubated with non-COVID-19 serum (Fig. 4).
COVID-19 environment induces lipid peroxidation. We evaluated lipid peroxidation by measuring the level of malondialdehyde (MDA) in HUVEC lysate. Data are from triplicate experiments; *P < 0.001.
Then, we verified whether human endothelial cells were able to produce miR-145 and miR-885 and whether such production was regulated by COVID-19. We detected both miRNAs in the HUVEC lysate; interestingly, the levels of both miRNAs were reduced in response to COVID-19 serum exposure (Fig. 5, A and B). This phenomenon confirms that endothelial cells are able to actively release these miRNAs and that stress conditions can affect miR-145 and miR-885 production by endothelial cells.
Endothelial cells produce miR-145 and miR-885, and COVID-19 serum affects their levels. Quantification of miR-145 and miR-885 in HUVEC lysate, after incubation with the indicated sera for 24 h. Data are from triplicate experiments; *P < 0.001.
Discussion
One of the main findings of the present study is the identification of two miRNAs, miR-145 and miR-885, as potential predictors of thrombotic risk in patients with COVID-19. Indeed, miR-145 and miR-885 are significantly downregulated in COVID-19 patients with elevated circulating levels of D-dimer, and our correlation analysis confirms that miR-145 and miR-885 inversely correlate with D-dimer. Moreover, the downregulation of these miRNAs predicts mortality in patients with COVID-19. Indeed, the group of patients that died for COVID-19 exhibited lower levels of both miR-145 and miR-885 at baseline. These data unveil the clinically relevant predictor value of these miRNAs, also suggesting a causal implication of these molecules in determining higher thrombotic risk and mortality in patients with COVID-19. Consistently, these two miRNAs are implicated in the coagulation pathway; indeed, tissue factor (TF) has been identified as a direct target of miR-145 (Sahu et al., 2017), whereas miR-885 targets the von Willebrand Factor (vWF) (Zhang et al., 2019). In agreement with our findings, the downregulation of these miRNAs should promote higher levels of TF and vWF, thus evoking a prothrombotic state. By modulating these two factors, endothelial cells can regulate the activation of the coagulation cascade. One limitation of our study is that we did not determine the exact source of exosomes in our population; nevertheless, since endothelial dysfunction is a prominent feature of COVID-19, functionally contributing to the proinflammatory and prothrombotic state of the vasculature (Bikdeli et al., 2020), we speculate that at least one of the main sources of exosomes could be represented by endothelial cells, which indeed do express these miRNAs in normal conditions (Santulli, 2016). To test this hypothesis, we performed in vitro experiments in human endothelial cells. Specifically, we set an in vitro model in which we verified the expression of cofactors needed for the internalization of SARS-CoV-2 in host cells, and we explored endothelial stress responses to a “COVID-like environment” represented by serum from patients with COVID-19. We found that COVID-19 serum (collected on hospital admission) induces endothelial damage, including cell apoptosis and alterations of a specialized endothelial feature like angiogenic capacity. These findings are in line with numerous reports from us and others supporting the endothelial involvement in COVID-19 clinical manifestations (Libby and Lüscher, 2020; Gu et al., 2021; Teuwen et al., 2020; Fiorentino et al., 2021; Gambardella and Santulli, 2021; Mesquida et al., 2021; Mone et al., 2021; Perea Polak et al., 2021; Qin et al., 2021; Schmaier et al., 2021; Yin et al., 2021; Kelliher et al., 2022; Mone et al., 2022; Otifi and Adiga, 2022; Robles et al., 2022).
One of the main new findings unveiled here is that the dysfunctional endothelium could participate in thrombotic manifestations by changing its miRNA profile. As proposed here, endothelial cells physiologically produce miR-145 and miR-885 to target vWF and TF, blocking their release and controlling coagulation. Indeed, we observed that endothelial cells are able to produce these miRNAs, and under stress conditions (i.e., when exposed to COVID-19 serum), their levels significantly decrease. Consistently, in patients in which these adaptive mechanisms are compromised, the resultant lower availability of miR-145 and miR-885 predisposes to a detrimental prothrombotic status, denoted by high D-dimer, and eventually high mortality. Our data in COVID-19 patients combined with in vitro experiments are highly suggestive of this view. Our report further exposes the endothelium as a central player in orchestrating the adaptative and maladaptive response to SARS-CoV-2 infection, acting as a main trigger of thrombotic manifestations.
Consistent with these findings, the importance of miRs and other noncoding RNAs in the management of patients with COVID-19 has been emphasized by several investigators (Amini-Farsani et al., 2021; Battaglia et al., 2021; Dash et al., 2021; Farr et al., 2021; Lukiw, 2021; Narożna and Rubiś, 2021; Plowman and Lagos, 2021; Saha et al., 2021).
Our data can be also useful for the management and treatment of Long COVID; in fact, the so-called “Long COVID-19 Syndrome”, indicating the set of disorders and clinical manifestations that remain or appear de novo months after COVID-19 infection and SARS-CoV-2 negativization, is emerging as the next challenge in biomedical research (Yan et al., 2021; Antoniou et al., 2022; Cattadori et al., 2022; Desai et al., 2022; Martínez-Salazar et al., 2022; Murray et al., 2022; Oikonomou et al., 2022; Thye et al., 2022). Henceforward, the deep understanding of the molecular mechanisms underlying the major complications of COVID-19 will allow us to intervene and thwart Long COVID.
Acknowledgments
The authors thank Dr. Varzideh for insightful discussion.
Authorship Contributions
Participated in research design: Gambardella, Santulli.
Conducted experiments: Gambardella, Kansakar, Messina, Jankauskas, Marfella, Maggi, Mone, Paolisso, Sorriento.
Contributed new reagents or analytic tools: Gambardella, Kansakar, Messina, Jankauskas, Marfella, Maggi, Mone, Paolisso, Sorriento.
Performed data analysis: Gambardella, Kansakar, Santulli.
Wrote or contributed to the writing of the manuscript: Gambardella, Kansakar, Sorriento, Santulli.
Footnotes
- Received March 14, 2022.
- Accepted May 9, 2022.
The Santulli’s Laboratory is supported in part by National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [Grant R01-DK123259] and [Grant R01-DK033823] and National Heart, Lung, and Blood Institute (NHLBI) [Grant R01-HL159062], [Grant R01-HL146691], and [Grant T32-HL144456] (to G.S.); the Diabetes Action Research and Education Foundation (to G.S.); and the Monique Weill-Caulier and Irma T. Hirschl Trusts (to G.S.). J.G. and S.S.J. are supported by a postdoctoral fellowship of the American Heart Association [AHA-20POST35211151] and [AHA-21POST836407], respectively.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abbreviations
- COVID-19
- coronavirus disease 2019
- HUVEC
- human umbilical vein endothelial cell
- miRNA
- microRNA
- TF
- tissue factor
- vWF
- von Willebrand factor
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
