Association between immune checkpoint inhibitor-induced myocarditis and concomitant use of thiazide diuretics.

Although an association has been reported between diuretics and myocarditis, it is unclear whether the risk of immune checkpoint inhibitor (ICI)-induced myocarditis is affected by concomitant diuretics. Thus, the aim of this work was to evaluate the impact of concomitant diuretics on ICI-induced myocarditis. This cross-sectional study used disproportionality analysis and a pharmacovigilance database to assess the risk of myocarditis with various diuretics in patients receiving ICIs via the analysis of data entered into the VigiBase database through December 2022. Multiple logistic regression analysis was performed to identify risk factors for myocarditis in patients who received ICIs. A total of 90 611 patients who received ICIs, including 975 cases of myocarditis, were included as the eligible dataset. A disproportionality in myocarditis was observed for loop diuretic use (reporting odds ratio 1.47, 95% confidence interval [CI] 1.02-2.04, P = .03) and thiazide use (reporting odds ratio 1.76, 95% CI 1.20-2.50, P < .01) in patients who received ICIs. The results of the multiple logistic regression analysis showed that the use of thiazides (odds ratio 1.67, 95% CI 1.15-2.34, P < .01) was associated with an increased risk of myocarditis in patients who received ICIs. Our findings may help to predict the risk of myocarditis in patients receiving ICIs.

Serum amyloid A-induced blood-brain barrier dysfunction associated with decreased claudin-5 expression in rat brain endothelial cells and its inhibition by high-density lipoprotein in vitro.

The blood-brain barrier (BBB) is the multicellular interface located between the peripheral circulation and the brain parenchyma. BBB dysfunction is reported in many CNS diseases, such cognitive impairment, depression, Alzheimer's disease (AD), and multiple sclerosis (MS). Emerging evidence indicates that liver-derived inflammatory mediators are upregulated in neurological diseases with BBB dysfunction. Serum amyloid A (SAA), an acute phase protein secreted by hepatocytes, could be a candidate inflammatory signaling molecule transmitted from the liver to the brain; however, its contribution to BBB dysfunction is poorly understood. The present study aimed to elucidate the involvement of SAA in BBB impairment in an in vitro BBB model using rat brain microvascular endothelial cells (RBECs). We demonstrated that Apo-SAA significantly decreased transendothelial electrical resistance (TEER) and increased sodium fluorescein (Na-F) permeability in RBEC monolayers. Apo-SAA also decreased claudin-5 expression levels in RBECs. Furthermore, the Apo-SAA-mediated impairment of the BBB with decreased claudin-5 expression was inhibited by the addition of a high-density lipoprotein (HDL) related to SAA in plasma. These findings suggest that HDL counteracts the effects of SAA on BBB function. Therefore, the functional imbalance between SAA and HDL may induce BBB impairment, thereby triggering development of neuroinflammation. SAA could be a significant endogenous mediator in the liver-to-brain inflammation axis.

Oligodendrocytes upregulate blood-brain barrier function through mechanisms other than the PDGF-BB/PDGFRα pathway in the barrier-tightening effect of oligodendrocyte progenitor cells.

White matter lesions are associated with impairment of the blood-brain barrier (BBB), an essential component of the cerebrovasculature. The BBB allows the brain to maintain its highly specialized microenvironment by restricting entry of blood-borne substances including molecules that induce myelin damage. Accumulating evidence suggests that interactions between brain endothelial cells and neighboring cells, including oligodendrocyte progenitor cells (OPCs), are required for the induction and maintenance of BBB function. Here, we compared the ability of OPCs and oligodendrocytes to modulate BBB integrity using co-cultures of rat brain endothelial cells with OPCs or oligodendrocytes. We found that OPCs lowered the brain endothelial permeability to sodium fluorescein, and this enhancement of BBB function was prevented by treatment with AG1296 (a PDGFRα inhibitor). Oligodendrocytes also enhanced BBB integrity. Pharmacological inhibition of PDGFRα did not affect the oligodendrocyte-induced BBB facilitation. These data indicate that oligodendrocytes enhance BBB integrity through pathways other than PDGF-BB/PDGFRα signaling triggered by the brain endothelial cell-derived PDGF-BB. Therefore, our findings suggest that oligodendrocytes constitutively support BBB integrity through soluble factors. Crosstalk between brain endothelial cells and oligodendrocytes could play a facilitatory role in maintaining BBB integrity in the white matter.

Oncostatin-M-Reactive Pericytes Aggravate Blood-Brain Barrier Dysfunction by Activating JAK/STAT3 Signaling In Vitro.

Oncostatin M (OSM) is a cytokine of the interleukin (IL)-6 family members. It induces blood-brain barrier (BBB) dysfunction by activating Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) 3 pathways in brain endothelial cells. Brain pericytes located around microvessels are one of the BBB constituents. Pericytes work as a boundary surface between the blood circulation and brain parenchyma, and their functions are altered under pathophysiological conditions, leading to BBB dysregulation. However, it remains unknown whether pericytes are associated with OSM-induced BBB dysfunction. We demonstrated that pericyte exposure to OSM (100 ng/mL) elevated phosphorylation of STAT3, a main OSM signaling pathway, and that pericytes expressed OSM receptors (OSMRs) including OSMRß and glycoprotein 130. These results suggest that pericytes are able to respond to OSM. To determine the effects of OSM-reactive pericytes on BBB functions, rat brain endothelial cell (RBEC) monolayers were cultured with OSM-treated pericytes. The presence of pericytes exposed to 100 ng/mL of OSM for 48 h aggravated both the elevated permeability to sodium fluorescein and the lowered transendothelial electrical resistance which were induced by OSM in RBECs. This OSM-reactive pericyte-induced aggravation of lowered RBEC barrier function was reversed by ruxolitinib, a JAK inhibitor. These findings suggest that activated JAK/STAT3 signaling in pericytes contributes to OSM-produced BBB breakdown. Thus, OSM-reactive pericytes may have to be considered a characteristic machinery in the formation and progression of BBB breakdown under pathological conditions associated with increased OSM levels.

Monomeric α-synuclein induces blood-brain barrier dysfunction through activated brain pericytes releasing inflammatory mediators in vitro.

Blood-brain barrier (BBB) disruption is often mediated by neuroinflammation, and occurs during various neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by loss of dopaminergic neurons and aggregated α-synuclein protein in inclusions known as Lewy bodies. Misfolded α-synuclein has been implicated in neurodegeneration and neuroinflammation through activation of microglia and astrocytes. Pericytes are a key cellular regulator of the BBB, although it is not known if they participate in α-synuclein-associated PD pathology. Here, we investigated the impact of pericytes on BBB integrity in response to α-synuclein using rat brain endothelial cells (RBECs) co-cultured with rat brain pericytes (RBEC/pericyte co-culture). In RBEC/pericyte co-cultures, α-synuclein added to the abluminal chamber (where pericytes were grown) significantly increased RBEC permeability to sodium fluorescein. In contrast, it had no marked effect when added to the luminal chamber. In the absence of pericytes, both luminal and abluminal addition of α-synuclein failed to affect permeability of the RBEC monolayer. α-Synuclein did not self-assemble in culture media within 24 h, suggesting that monomeric α-synuclein can disrupt the BBB by interacting with pericytes. We found that in response to α-synuclein, pericytes, but not RBECs, released interleukin (IL)-1ß, IL-6, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, and matrix metalloproteinase-9 (MMP-9). α-Synuclein did not affect platelet-derived growth factor (PDGF)-BB release from RBECs and PDGF receptor-ß expression in pericytes. These results suggest that pericytes are more sensitive to monomeric α-synuclein than RBECs regarding release of various inflammatory cytokines/chemokines and MMP-9. Thus, monomeric α-synuclein-activated pericytes may contribute to BBB breakdown in patients with PD.

Activation of the α7 nicotinic acetylcholine receptor upregulates blood-brain barrier function through increased claudin-5 and occludin expression in rat brain endothelial cells.

The blood-brain barrier (BBB) is formed by brain endothelial cells (BECs) and regulates brain homeostasis by restricting the entry of blood-borne substances into the brain. Recent in vivo studies have shown that administration of nicotinic acetylcholine receptor (nAChR) agonists protects against BBB disruption and neuroinflammation induced by stroke and traumatic brain injury through the systemic cholinergic anti-inflammatory pathway. In the present study, we focused on the nAChRs expressed on BECs rather than those widely expressed in the central nervous system and peripheral tissues, and examined whether activation of the nAChRs on BECs facilitates BBB function. We used primary cultures of rat brain endothelial cells to evaluate brain endothelial permeability and tight junction (TJ)-related protein expression after a 24-h exposure to PHA543613 (a selective α7 nAChR agonist) or 5-iodo-A-85380 (a selective α4ß2 nAChR agonist). We found that PHA543613 decreased sodium fluorescein permeability and increased the expression levels of claudin-5 and occludin, key TJ components. In contrast, 5-iodo-A-85380 had no effect on brain endothelial permeability or TJ protein expression. These findings suggest that the selective activation of α7 nAChRs on BECs has a specific role in upregulating BBB properties through increased claudin-5 and occludin expression.

Oncostatin M-induced blood-brain barrier impairment is due to prolonged activation of STAT3 signaling in vitro.

Oncostatin M (OSM) is a member of the interleukin (IL)-6 family cytokines. We previously demonstrated that OSM induces blood-brain barrier (BBB) impairment. However, functional characterization of IL-6 family cytokines in BBB regulation and the cytokine-related intracellular signaling pathway remain unclear. In this study, we demonstrate that among IL-6 family cytokines, including IL-6 and leukemia inhibitory factor (LIF), OSM is the most potent molecule for inducing BBB dysfunction via prolonged activation of signal transducer and activator of transcription (STAT) 3 following Janus-activated kinase (JAK) activation. OSM but not IL-6 and LIF (100 ng/mL for 24 hours) markedly produced increased sodium fluorescein permeability and decreased transendothelial electrical resistance in rat brain endothelial cell (RBEC) monolayers. This OSM-induced BBB dysfunction was accompanied by decreased levels of claudin-5 expression in RBECs, which were ameliorated by JAK inhibitor. We examined the time-course of STAT3 phosphorylation in RBECs treated with OSM, IL-6, and LIF. OSM upregulated STAT3 phosphorylation levels during a 24 hours period with a peak at 10 minutes. While IL-6 and LIF transiently increased phosphorylated STAT3 at 10 minutes after addition, this phosphorylation decreased during the period from 1 to 24 hours after addition. These findings suggest that OSM-induced sustained increases in STAT3 phosphorylation levels largely contribute to BBB impairment. Thus, elevated OSM levels and activation of brain endothelial JAK/STAT3 signaling pathway under pathological conditions should be considered as a possible hallmark for induction and development of BBB impairment.

Contribution of thrombin-reactive brain pericytes to blood-brain barrier dysfunction in an in vivo mouse model of obesity-associated diabetes and an in vitro rat model.

Diabetic complications are characterized by the dysfunction of pericytes located around microvascular endothelial cells. The blood-brain barrier (BBB) exhibits hyperpermeability with progression of diabetes. Therefore, brain pericytes at the BBB may be involved in diabetic complications of the central nervous system (CNS). We hypothesized that brain pericytes respond to increased brain thrombin levels in diabetes, leading to BBB dysfunction and diabetic CNS complications. Mice were fed a high-fat diet (HFD) for 2 or 8 weeks to induce obesity. Transport of i.v.-administered sodium fluorescein and 125I-thrombin across the BBB were measured. We evaluated brain endothelial permeability and expression of tight junction proteins in the presence of thrombin-treated brain pericytes using a BBB model of co-cultured rat brain endothelial cells and pericytes. Mice fed a HFD for 8 weeks showed both increased weight gain and impaired glucose tolerance. In parallel, the brain influx rate of sodium fluorescein was significantly greater than that in mice fed a normal diet. HFD feeding inhibited the decline in brain thrombin levels occurring during 6 weeks of feeding. In the HFD fed mice, plasma thrombin levels were significantly increased, by up to 22%. 125I-thrombin was transported across the BBB in normal mice after i.v. injection, with uptake further enhanced by co-injection of unlabeled thrombin. Thrombin-treated brain pericytes increased brain endothelial permeability and caused decreased expression of zona occludens-1 (ZO-1) and occludin and morphological disorganization of ZO-1. Thrombin also increased mRNA expression of interleukin-1ß and 6 and tumor necrosis factor-α in brain pericytes. Thrombin can be transported from circulating blood through the BBB, maintaining constant levels in the brain, where it can stimulate pericytes to induce BBB dysfunction. Thus, the brain pericyte-thrombin interaction may play a key role in causing BBB dysfunction in obesity-associated diabetes and represent a therapeutic target for its CNS complications.

Role of thrombin-PAR1-PKCθ/δ axis in brain pericytes in thrombin-induced MMP-9 production and blood-brain barrier dysfunction in vitro.

Thrombin, an essential component in the coagulation cascade, participates in the pathogenesis of brain diseases, such as ischemic stroke, intracerebral hemorrhage, Alzheimer's disease and Parkinson's disease through blood-brain barrier (BBB) dysfunction. It is thought that the thrombin-matrix metalloproteinase (MMP)-9 axis is an important process in the pathogenesis of neurovascular disease, such as BBB dysfunction. We recently reported that brain pericytes are the most MMP-9-releasing cells in response to thrombin stimulation among the BBB-constituting cells. This thrombin-induced MMP-9 release is partially due to protease-activated receptor (PAR1), one of the specific thrombin receptors. Then, we evaluated the intracellular signaling pathways involved in MMP-9 release and the contribution of thrombin-reactive brain pericytes to BBB dysfunction. PKC activator evoked MMP-9 release from brain pericytes. The thrombin-induced MMP-9 release was inhibited by U0126, LY294002, Go6976, and Go6983. However, Go6976 decreased phosphorylation levels of PKCθ and Akt, and Go6983 decreased phosphorylation levels of PKCδ and extracellular signal-regulated kinase (ERK). Additionally, treatment of pericytes with thrombin or PAR1-activating peptide stimulated PKCδ/θ signaling. These substances impaired brain endothelial barrier function in the presence of brain pericytes. Brain pericytes function through two independent downstream signaling pathways via PAR1 activation to release MMP-9 in response to thrombin - the PKCθ-Akt pathway and the PKCδ-ERK1/2 pathway. These pathways participate in PAR1-mediated MMP-9 release from pericytes, which leads to BBB dysfunction. Brain pericytes and their specific signaling pathways could provide novel therapeutic targets for thrombin-induced neurovascular diseases.

Nifedipine prevents sodium caprate-induced barrier dysfunction in human epidermal keratinocyte cultures.

Tight junctions (TJs) of the epidermis play an important role in maintaining the epidermal barrier. TJ breakdown is associated with skin problems, such as wrinkles and transepidermal water loss (TEWL). Clinical studies have reported that topical nifedipine is effective in reducing the depth of wrinkles and improving TEWL. However, it remains unknown whether nifedipine influences the TJ function in the epidermis. In the present study, we investigated the effect of nifedipine on epidermal barrier dysfunction in normal human epidermal keratinocytes (NHEKs) treated with sodium caprate (C10), a TJ inhibitor. Nifedipine reversed the C10-decreased transepithelial electrical resistance values as a measure of disruption of the epidermal barrier. Immunocytochemical observations revealed that nifedipine improved the C10-induced irregular arrangement of claudin-1, a key protein in TJs. Taken together, these findings suggest that nifedipine prevents epidermal barrier dysfunction, at least in part, by reconstituting the irregular claudin-1 localization at TJs in C10-treated NHEKs.

Brain pericytes are the most thrombin-sensitive matrix metalloproteinase-9-releasing cell type constituting the blood-brain barrier in vitro.

In the acute phase of intracerebral hemorrhage (ICH), hemorrhagic transformation and brain edema are associated with blood-brain barrier (BBB) disruption. Elevated levels of thrombin, a coagulation factor, contribute to the development of brain edema during ICH through matrix metalloproteinase (MMP)-9 production. Thrombin directly induces a variety of cellular responses through its specific receptors known as protease-activated receptors (PARs). However, it remains unclear which cell types constituting the BBB mainly produce MMP-9 in response to thrombin. Here, we compared the MMP-9 release induced by thrombin using primary cultures of rat brain microvascular endothelial cells, astrocytes, and pericytes. Brain pericytes exhibited the highest levels of MMP-9 release due to thrombin stimulation among the BBB cells. The pattern of PAR mRNA expression in pericytes was characterized by high expression of PAR1 and moderate expression of PAR4. Heat-inactivated thrombin failed to stimulate pericytes to release MMP-9. A selective PAR1 inhibitor SCH79797 blocked the thrombin-induced MMP-9 release from pericytes. These findings suggest that both PAR1 and PAR4 mediate thrombin-induced MMP-9 release from pericytes. The present study raises the possibility that brain pericytes could play a pivotal role as a highly thrombin-sensitive and MMP-9-producing cell type at the BBB in brain damage including ICH.