Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases

Abstract

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction–related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2–induced endothelial hyperpermeability, suggesting that SARS-CoV-2–induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2–treated mice. These results indicate that Robo4 expression–increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

1. INTRODUCTION

When a pathogen infects the body, it multiplies, activates vascular endothelial cells (ECs) and immune cells, and induces inflammatory cytokine production. Inflammatory cytokines, such as tumor necrosis factor α (TNFα), induce vascular hyperpermeability. These processes are part of the body’s defense system, which efficiently recruits immune cells to pathogen invasion sites to rapidly eliminate pathogens. However, in severe infections, these processes are excessively enhanced, leading to fatal pathological conditions (Fig. 1). To date, several highly effective drugs have been developed, including antipathogenic drugs, steroids, and anti-inflammatory cytokine drugs that suppress pathogen propagation, immune cell activation, and inflammatory cytokine functions. However, even these drugs are unable to completely control the pathology and mortality of severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, it is important to develop novel drugs that can suppress vascular hyperpermeability and treat severe infectious diseases.

Fig. 1. Processes That Induce Severe Infections and Therapeutic Drugs for Each Process

Vascular permeability is regulated by ECs, which line the lumen of blood vessels. Endothelial permeability is regulated by cell–cell junctions. Adherens junctions are regulated by cadherins and nectins, whereas tight junctions are regulated by occludins, claudins, and junctional adhesion molecules.^1,2) Among these proteins, vascular endothelial cadherin (VE-cadherin) and Claudin-5 (CLDN5), which are specifically expressed in ECs, are well-known regulators of endothelial permeability (Fig. 2). VE-cadherin is important for maintaining vascular integrity and permeability.³⁾ CLDN5 controls the tight sealing of ECs in the blood–brain barrier. CLDN5 is a well-studied regulator of the blood–brain barrier that inhibits the transfer of small molecules across the vasculature of the brain.⁴⁾ However, the functions of CLDN5 in other organs remain poorly understood. Under normal conditions, VE-cadherin and CLDN5 contribute to cell–cell junctions and maintain low permeability.^1–3) However, under inflammatory conditions induced by pathogens, these adhesions are weakened, and vascular permeability increases. Vascular hyperpermeability in severe infectious diseases can lead to severe, and sometimes fatal, organ dysfunction.

Fig. 2. Endothelial Cell Junction Proteins Contributing to Vascular Permeability Regulation

Many patients with severe COVID-19 experience acute respiratory distress syndrome, pneumonia, and pulmonary edema, which are induced by vascular hyperpermeability in the lungs.⁵⁾ In addition, the induction of cytokine storms, infiltration of immune cells, and vascular leakage in patients with severe COVID-19 are regulated by ECs.^6,7) However, it is unclear how ECs regulate endothelial hyperpermeability and inflammation in patients with COVID-19. Therefore, we analyzed the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on endothelial junctions and permeability.

2. ENDOTHELIAL BARRIER DISRUPTION IN COVID-19 MODELS

We analyzed the effect of SARS-CoV-2 on vascular permeability using an airway-on-a-chip^8,9) (Fig. 3). The airway-on-a-chip is a co-culture system consisting of two channels seeded with pulmonary microvascular ECs and airway epithelial cells that mimic the respiratory and pulmonary microenvironments. Using this chip, we investigated how SARS-CoV-2 invades the blood vessels of respiratory organs.¹⁰⁾ When the airway channel was treated with SARS-CoV-2, the virus infected epithelial cells and multiplied. Subsequently, an increase in SARS-CoV-2 was observed in the vascular channels, indicating that this chip infection model could simulate SARS-CoV-2 invasion into blood vessels. RNA-seq analysis of gene expression patterns in the ECs of the chip showed that SARS-CoV-2 suppressed the expression of CLDN5, which is responsible for cell–cell adhesion (tight junctions) between ECs. In contrast, no changes were observed in the expression of VE-cadherin, which contributes to the formation of adherens junctions. Immunostaining analysis also showed a SARS-CoV-2–mediated decrease in CLDN5 expression in ECs. In addition, SARS-CoV-2 disrupted VE-cadherin–mediated junctions and generated gaps between cells that are sufficiently large for viral passage. This indicates that SARS-CoV-2 induces excessive vascular hyperpermeability via endothelial barrier disruption by suppressing CLDN5 expression.

Fig. 3. SARS-CoV-2 Infection Model Using Airway-on-a-Chip

Airway-on-a-chip contains airway and vascular channels, in which epithelial and endothelial cells were seeded, respectively, to mimic respiratory organs.

To investigate the physiological significance of the SARS-CoV-2–mediated CLDN5 downregulation, we analyzed CLDN5 functions in mice.¹⁰⁾ When mice were intravenously injected with an anti-CLDN5 antibody that binds to the extracellular domain of CLDN5 and inhibits its function, severe vascular leakage was induced, specifically in the lungs, leading to pulmonary edema. These results suggest that the decrease in SARS-CoV-2–induced CLDN5 expression contributes to endothelial barrier disruption and the induction of pulmonary edema.

To investigate the therapeutic strategy against SARS-CoV-2–mediated endothelial barrier disruption, we analyzed whether enhanced CLDN5 expression alleviates endothelial barrier disruption induced by SARS-CoV-2.¹⁰⁾ Expression vector-mediated CLDN5 expression suppressed SARS-CoV-2–induced endothelial barrier disruption and induced inflammatory gene expression in airway-on-a-chip. Fluvastatin was identified in a search for small molecules that increase CLDN5 expression. Fluvastatin treatment enhanced endothelial CLDN5 expression and suppressed SARS-CoV-2–induced endothelial barrier disruption, viral passage, and inflammatory gene expression in the airway-on-a-chip. These results indicate that enhanced CLDN5 expression can suppress SARS-CoV-2–induced endothelial barrier disruption and inflammation.

Finally, we analyzed the endothelial barrier disruption activity of SARS-CoV-2 variants.¹⁰⁾ The results showed that the δ strain, which has been reported to induce the most severe pathology of COVID-19, induces endothelial barrier disruption most strongly. This suggests that the endothelial barrier disruption activity of SARS-CoV-2 variants correlates with the severity of COVID-19 pathology.

Collectively, our findings show that SARS-CoV-2 disrupts the endothelial barrier by suppressing CLDN5 expression, leading to vascular hyperpermeability and edema in the lungs. In addition, endothelial barrier disruption can be suppressed by drugs promoting CLDN5 expression (Fig. 4). These findings suggest that decreasing vascular hyperpermeability may be a novel therapeutic strategy for suppressing severe infectious diseases.

Fig. 4. SARS-CoV-2–Induced Vascular Hyperpermeability and Potential Therapeutic Strategy

3. SUPPRESSION OF VASCULAR PERMEABILITY VIA THE ENDOTHELIAL-SPECIFIC PROTEIN, ROUNDABOUT4 (ROBO4)

Our results suggest that enhancing CLDN5 expression could be a potential strategy for suppressing endothelial barrier disruption in severe infectious diseases. We then focused on the endothelial-specific protein Robo4^11,12) that suppresses vascular permeability by stabilizing ECs and ameliorating severe infectious diseases in mouse models.¹³⁾ Robo4 has also been shown to regulate inflammatory cytokine production.¹⁴⁾ However, the detailed mechanism by which Robo4 suppresses infectious and inflammatory diseases is unclear as previous reports have proposed multiple and partially controversial mechanisms.^15–18) To investigate the mechanism by which Robo4 suppresses vascular permeability in inflammatory and infectious diseases, we analyzed Robo4 function in Robo4 knockout mice injected with lipopolysaccharide (LPS).¹⁹⁾ In this model, LPS activates ECs and immune cells, thereby inducing the production of inflammatory cytokines. The cytokines, including TNFα, induce vascular hyperpermeability and fatal pathologies. Robo4 knockout in LPS-injected mice increases vascular leakage in multiple organs, including the lungs, heart, and small intestine, and promotes mortality. These results indicated that endothelial Robo4 suppressed vascular permeability and reduced mortality in LPS-treated mice.

We then investigated the molecular mechanism by which Robo4 suppresses vascular permeability. Under non-inflammatory conditions, ECs adhere to each other via VE-cadherin, and vascular permeability remains low. However, inflammatory stimuli such as TNFα induce vascular hyperpermeability by dissociating VE-cadherin–mediated interactions. To investigate the effect of Robo4 on endothelial permeability during inflammation, we measured transendothelial electrical resistance (TEER) using human primary ECs (human umbilical vein ECs).¹⁹⁾ TNFα treatment decreased TEER in ECs, indicating increased endothelial permeability. Small interfering RNA (siRNA)-mediated Robo4 knockdown enhanced this permeability. In contrast, viral vector–mediated Robo4 overexpression suppressed the TNFα-induced endothelial hyperpermeability. These results indicate that Robo4 suppresses TNFα-induced endothelial hyperpermeability and that endothelial permeability under inflammatory condition can be manipulated both positively and negatively by controlling Robo4 expression levels in ECs.

We also analyzed the effect of Robo4 on VE-cadherin localization.¹⁹⁾ Under normal conditions, VE-cadherin is localized at the cell–cell junctions between ECs. However, TNFα treatment induced VE-cadherin dissociation and translocation into the cells. siRNA-mediated Robo4 knockdown enhanced this VE-cadherin redistribution induced by TNFα. Conversely, viral vector–mediated Robo4 overexpression suppressed the redistribution of VE-cadherin. Collectively, these results indicate that Robo4 suppresses vascular permeability by stabilizing VE-cadherin localization at cell–cell junctions, even under inflammatory conditions.

To further investigate the molecular mechanisms underlying Robo4-mediated suppression of endothelial permeability, we analyzed the Robo4 domain, which contributes to permeability suppression.¹⁹⁾ A Robo4 mutant lacking N-terminus regions, as well as full-length Robo4, was found to suppress the TNFα-induced VE-cadherin redistribution; however, a mutant lacking C-terminal regions did not induce this effect, indicating that Robo4 suppresses permeability via the C-terminal region. To purify proteins that interact with the Robo4 C-terminus, we expressed FLAG-tagged Robo4 in ECs and purified Robo4-interacting proteins by immunoprecipitation. Mass spectrometric analysis identified TNF receptor-associated factor 7 (TRAF7). Co-immunoprecipitation assays using Robo4 mutants showed that TRAF7 interacted with the C-terminal region of Robo4.

TRAF7 belongs to the TRAF family of proteins that regulate the inflammatory and apoptotic signaling pathways.^20–23) In general, TRAF proteins transduce signals via the TRAF-C domain. However, TRAF7 is the only member that contains the WD40 domain instead of the TRAF-C domain, which interacts with TNF receptors.²⁴⁾ Although TRAF7 has been shown to function as a E3-ubiquitin ligase and regulate mitogen-activated protein kinase kinase kinase 3 and TNFα signaling in cell lines,^20,21) TRAF7 functions remain unknown, especially in normal cells such as ECs. To investigate potential functions of TRAF7 in ECs, we analyzed the subcellular localization of Robo4 and TRAF.¹⁹⁾ Since Robo4 has been shown to be localized in both the cell membrane and cytoplasm,²⁵⁾ we performed immunofluorescent staining to determine where Robo4 interacts with TRAF7. In cells expressing either Robo4 or TRAF7, Robo4 was localized near the nucleus, whereas TRAF7 was localized in small cytoplasmic particles. When Robo4 and TRAF7 were co-expressed, they co-localized near the nucleus. This colocalization was not observed in the Robo4 mutant, which lacks the C-terminal region. These results show that TRAF7 is recruited to the vicinity of the nucleus by Robo4 via its C-terminal region.

We evaluated the effect of TRAF7 on endothelial permeability.¹⁹⁾ TRAF7 knockdown mediated by siRNA enhanced VE-cadherin redistribution and endothelial hyperpermeability induced by TNFα. Conversely, TRAF7 overexpression suppressed endothelial redistribution and hyperpermeability, indicating that TRAF7 suppressed endothelial permeability. Because TRAF7 has a function similar to that of Robo4, we analyzed whether Robo4 or TRAF7 was the main regulator of permeability. TRAF7 overexpression under Robo4 knockdown condition suppressed TNFα-induced endothelial hyperpermeability. However, Robo4 overexpression with TRAF7 knockdown did not suppress hyperpermeability. This indicates that TRAF7 is the main regulator of endothelial permeability and that Robo4 enhances TRAF7. Collectively, Robo4 suppressed vascular hyperpermeability by interacting with TRAF7, leading to decreased mortality in LPS-injected mice (Fig. 5).

Fig. 5. Robo4-TRAF7 Complex–Mediated Suppression Mechanism of Endothelial Permeability

Robo4 interacts with TRAF7 and suppresses inflammatory cytokine (TNFα)–induced VE-cadherin dissociation.

4. ROBO4 OVEREXPRESSION SUPPRESSES PATHOLOGIES IN LPS-INJECTED MICE

Our data showed that manipulation of Robo4 expression alters endothelial permeability during inflammation and that Robo4 knockout exacerbated pathology in LPS-injected mice. Based on this information, we hypothesized that the upregulation of Robo4 expression suppresses vascular permeability and alleviates severe infectious diseases. To test this hypothesis, we generated tamoxifen-induced endothelial-specific Robo4 overexpression mice (Robo4^iEC mice).²⁶⁾ When Robo4^iEC mice were injected with tamoxifen, Robo4 expression increased 2–3 times in organs. Robo4^iEC mice injected with LPS exhibit decreased vascular leakage into the lungs and decreased mortality. These results indicate that upregulation of Robo4 expression suppresses vascular permeability and alleviates sepsis and suggest that drugs that regulate Robo4 expression can regulate vascular permeability and pathologies in severe infectious diseases.

5. SMALL MOLECULE DEVELOPMENT TO REGULATE ROBO4 EXPRESSION

To develop small molecules that control vascular permeability, we searched for compounds that regulate Robo4 expression. We previously demonstrated that Robo4 expression is regulated by the 3 kb human Robo4 promoter.²⁷⁾ Several transcription factors, such as GA-binding protein (GABP),^27,28) SP1,²⁷⁾ AP1,²⁹⁾ nuclear factor-kappaB (NF-κB),³⁰⁾ and ETV2,³¹⁾ bind to and activate the promoter. GABP binds to the proximal promoter and is essential for promoter activity.²⁸⁾ In addition, we showed that Robo4 expression is regulated by epigenetic mechanisms. CpG sequences in the Robo4 proximal promoter are differentially methylated in EC and non-ECs.³²⁾ EC-specific non-methylated promoters regulated by ETV2 induces endothelial-specific Robo4 expression.³¹⁾ We also found that histone deacetylase (HDAC) inhibitors strongly suppress Robo4 expression.³³⁾ Therefore, we focused on one inhibitor, MS-275, as a model molecule to suppress Robo4 expression.

An analysis of the mechanism by which MS-275 suppresses Robo4 expression revealed that MS-275 reduces GABP expression via HDAC3 inhibition, which in turn reduces Robo4 expression.³³⁾ Analysis of the effect of MS-275 on vascular permeability showed that MS-275 decreased TEER and enhanced the migration of melanoma cells via an endothelial monolayer. Oral administration of MS-275 to mice enhanced Evans blue leakage and extravasation of melanoma cells into the lungs. These results indicate that MS-275, a suppressor of Robo4 expression can enhance vascular permeability.

6. SEARCH FOR MOLECULES THAT ENHANCE ROBO4 EXPRESSION

To further identify small molecules that regulate Robo4 expression, we investigated the signaling pathways that regulate Robo4 expression using library screening.²⁶⁾ Two EC lines stably transfected with either the Robo4 promoter-reporter (luciferase) or SV-40 (control) promoter-reporter sequences were generated. Using these cells, we screened compound libraries and identified compounds that regulated the activity of the Robo4 promoter but not the control promoter. Among the identified compounds that suppresses Robo4 expression, we found SB525334, which is known to inhibit the transforming growth factor (TGF)-β receptor, ALK5. Because TGF-β–mediated ALK5 activation induces activation of transcription factors SMAD2/3 and their downstream gene expression, we hypothesized that TGF-β-ALK5-SMAD2/3 pathways increase Robo4 expression. Indeed, siRNA-mediated SMAD2/3 knockdown decreased Robo4 expression in ECs, and TGF-β treatment enhanced Robo4 expression in ECs cultured in the Matrigel. These findings indicate that that the TGF-β-ALK5-SMAD2/3 pathway positively regulates Robo4 expression.

Previous reports indicate that the BMP9-ALK1-SMAD1/5 pathway functions in competition with the TGF-β-ALK5-SMAD2/3 pathway. Therefore, we hypothesized that BMP9-ALK1-SMAD1/5 signaling would suppress Robo4 expression. Consistently, BMP9 treatment suppressed Robo4 expression in ECs. This suppression was abolished by siRNA-mediated SMAD1/5 knockdown or treatment with K02288, an inhibitor of the BMP9 receptor, ALK1. These results indicate that the BMP9-ALK1-SMAD1/5 pathway negatively regulates Robo4 expression, suggesting that the ALK1 inhibitor, K02288, is a potential molecule that increases Robo4 expression (Fig. 6).

Fig. 6. Regulation Mechanisms of Robo4 Expression by ALK-SMAD Pathways

Robo4 expression is regulated positively and negatively by ALK5-SMAD2/3 and ALK1-SMAD1/5 signaling, respectively.

7. EFFECTS OF THE ALK1 INHIBITOR IN INFECTIOUS DISEASE MODELS

Because ALK1 is predominantly highly expressed in lung ECs, we investigated the effects of ALK1 inhibitors on mouse lungs.²⁶⁾ Intravenous injection of an ALK1 inhibitor increased Robo4 expression in the lung tissue. The ALK1 inhibitor suppresses Evans blue leakage and melanoma cell extravasation in the lungs of LPS-injected mice. However, these ALK1 inhibitor–mediated protective effects were not observed in Robo4 knockout mice, indicating that ALK1 inhibitors suppress vascular hyperpermeability by increasing Robo4 expression. Similarly, the ALK1 inhibitor suppressed mortality in LPS-injected mice but not in Robo4 knockout mice, indicating that the ALK1 inhibitor ameliorated the pathological phenotype of LPS-injected mice in a Robo4-dependent manner.

We analyzed the effects of ALK1 inhibitors using SARS-CoV-2 infection models. Treatment of ECs with an ALK1 inhibitor in the airway-on-a-chip inhibited SARS-CoV-2–induced endothelial barrier disruption and viral migration. Furthermore, an ALK1 inhibitor suppressed lung injury and mortality in SARS-CoV-2–infected mice. The results showed that the ALK1 inhibitor suppressed endothelial barrier disruption and mortality in SARS-CoV-2 infection models (Fig. 4). Collectively, enhancement of Robo4 expression may be a novel therapeutic strategy for suppressing vascular hyperpermeability in severe infectious diseases and reducing the risk of mortality.

8. CONCLUSION AND PERSPECTIVES

Analyses using SARS-CoV-2 infection and LPS injection models indicated that endothelial barrier disruption, associated with vascular hyperpermeability, is an important process in the development of severe infectious diseases. In addition, enhanced CLDN5 and Robo4 expression has been shown to suppress vascular hyperpermeability and mortality in severe infectious diseases and endotoxemia models. Drugs targeting vascular permeability are expected to be useful therapeutic strategies for a wide range of infectious diseases because of their pathogen-independent mechanisms of action. Furthermore, synergistic effects can be expected when drugs are used in combination with existing drugs that target pathogens, immune cells, and inflammatory cytokines. Drugs that suppress vascular permeability may contribute to treating severe infectious diseases in the future for which vaccines or therapeutic drugs have not yet been developed.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science KAKENHI (23H02629 and 22K19377), Japan Agency for Medical Research and Development (JP22fk0108551, JP21fk0108432, JP23ama121052, and JP23ama121054), Takeda Science Foundation, Nippon Foundation–Osaka University Project for Infectious Disease Prevention, and Shionogi Infectious Disease Research Promotion.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2023 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

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