Increased levels of circulating cell-free DNA in COVID-19 patients with respiratory failure.

Cell-free DNA (cfDNA) is released from injured cells and aggravates inflammation. Patients with coronavirus disease (COVID-19) often develop pneumonia and respiratory failure, and require oxygen therapy (OT), including mechanical ventilation (MV). It remains unclear whether cfDNA predicts the risk of receiving OT or MV in COVID-19 patients. Therefore, we hypothesized that circulating cfDNA levels could reflect the severity of respiratory failure and determine a therapeutic approach for oxygenation in patients with COVID-19. We analyzed cfDNA levels in serum samples from 95 hospitalized patients with COVID-19 at Showa University Hospital (Tokyo, Japan). cfDNA levels were assessed by measuring the copy numbers of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) using quantitative real-time PCR (qPCR). Both cf-nDNA and cf-mtDNA levels were negatively correlated with adjusted SpO2 for FiO2 (SpO2/FiO2 ratio). Elevated cf-nDNA and cf-mtDNA levels were associated with the requirement for OT or MV during patient admission. Multivariate logistic regression analysis revealed that cf-nDNA and cf-mtDNA levels were independent risk factors for OT and MV. These results suggest that both serum cf-nDNA and cf-mtDNA could serve as useful early biomarkers to indicate the necessity of OT or MV in patients with COVID-19.

A Comprehensive Retrospective Study on the Mechanisms of Cyclic Mechanical Stretch-Induced Vascular Smooth Muscle Cell Death Underlying Aortic Dissection and Potential Therapeutics for Preventing Acute Aortic Aneurysm and Associated Ruptures.

Acute aortic dissection (AAD) and associated ruptures are the leading causes of death in cardiovascular diseases (CVDs). Hypertension is a prime risk factor for AAD. However, the molecular mechanisms underlying AAD remain poorly understood. We previously reported that cyclic mechanical stretch (CMS) leads to the death of rat aortic smooth muscle cells (RASMCs). This review focuses on the mechanisms of CMS-induced vascular smooth muscle cell (VSMC) death. Moreover, we have also discussed the potential therapeutics for preventing AAD and aneurysm ruptures.

The role of antipsychotics and other drugs on the development and progression of neuroleptic malignant syndrome.

Neuroleptic malignant syndrome (NMS) is a rare but serious and sometimes fatal complication in patients taking antipsychotic drugs, and its underlying mechanism still remains unclear. The pharmacotherapy for psychotic disorders is complicated and often involves a combination of two or more drugs, including drugs other than antipsychotics. In the present study, we used the Japanese Adverse Drug Event Report (JADER) database to broadly investigate the drugs associated with NMS, following their related pathways, as well as the drug-drug interactions (DDIs) in NMS. All analyses were performed using data from the JADER database from April 2004 to May 2022. Single-drug signals were evaluated using the reporting odds ratio (ROR) and proportional reporting ratio (PRR), and drug pathways were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG). DDIs were evaluated using the Ω shrinkage measure and Chi-square statistics models. All drugs associated with 20 or more NMS cases in the JADER database exhibited signals for NMS, including non-antipsychotics. Pathways associated with the drugs included the dopaminergic or serotonergic synapses related to antipsychotics. DDIs leading to NMS were confirmed for several drug combinations exhibiting single-drug signals. This study confirmed the significant association of various drugs, including non-psychotics, with NMS and suggested that various pathways related to these drugs may be involved in the progression of NMS. In addition, several combinations of these drugs were found to interact (DDI), increasing the risk of NMS, which suggests that appropriate caution should be taken when administering these drugs.

Analysis of Appendicitis Cases in the Japanese Adverse Drug Event Report (JADER) Database.

Appendicitis is one of the most common abdominal surgical emergencies worldwide; however, its causes remain poorly understood. The Japanese Adverse Drug Event Report (JADER) database is a spontaneous reporting system (SRS) that can be utilized to analyze the safety signals of adverse events. In this study, we investigated the association between drug use and the onset of appendicitis using the JADER database. We first used the reporting odds ratio (ROR) as the signal and found signals for appendicitis, perforated appendicitis, and complicated appendicitis for 23, 9, and 1 drug, respectively. To investigate the level of hazard over time in drug-associated appendicitis, the Weibull shape parameter ß was calculated using a Weibull plot, which revealed drug-dependent patterns for changes in the risk of appendicitis over time for the eight drugs. Furthermore, logistic regression analysis was performed to account for the influence of age, sex, and primary disease, and a significant association was detected between two drugs and appendicitis. Several types of drugs, such as antitumor, antirheumatic, and anti-inflammatory drugs, were included in our analyses; however, only clozapine, which is used for patients with schizophrenia, was commonly identified in these analyses. The resulting data suggest that certain drugs may be associated with appendicitis and may require adequate attention.

Upregulation of iNOS Protects Cyclic Mechanical Stretch-Induced Cell Death in Rat Aorta Smooth Muscle Cells.

Aortic dissection and aneurysm are associated with abnormal hemodynamic loads originating from hypertension. Our previous study demonstrated that cyclic mechanical stretch (CMS, mimicked hypertension) caused the death of rat aortic smooth muscle cells (RASMCs) in a mitogen activated-protein kinases (MAPKs)-dependent manner. The current study investigated the effects of inducible nitric oxide synthase (iNOS) on CMS-induced RASMC death. cDNA microarrays for CMS-treated RASMCs showed that iNOS expression levels were increased in response to CMS. Real-time polymerase chain reaction (PCR) analysis demonstrated that this increase was p38 MAPK (p38)-dependent. NO production was also increased. This increase could be inhibited by p38 and iNOS inhibitors. Thus, CMS-induced iNOS synthesized NO. CMS-induced cell death in RASMCs was increased by the iNOS inhibitor but abrogated by the long-acting NO donor DETA-NONOate. Increased iNOS expression was confirmed in the abdominal aortic constriction mouse model. Signal transducers and activators of transcription 1 (STAT1) was activated in stretched RASMCs, and iNOS expression and NO production were inhibited by the STAT1 inhibitor nifuroxazide. Our findings suggest that RASMCs were protected by iNOS from CMS-stimulated cell death through the STAT1 and p38 signal pathways independently.

Proliferative Pathways of Vascular Smooth Muscle Cells in Response to Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia (IH) and is a risk factor for cardiovascular diseases (e.g., atherosclerosis) and chronic inflammatory diseases (CID). The excessive proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the progression of atherosclerosis. Hypoxia-inducible factor-1 and nuclear factor-κB are thought to be the main factors involved in responses to IH and in regulating adaptations or inflammation pathways, however, further evidence is needed to demonstrate the underlying mechanisms of this process in VSMCs. Furthermore, few studies of IH have examined smooth muscle cell responses. Our previous studies demonstrated that increased interleukin (IL)-6, epidermal growth factor family ligands, and erbB2 receptor, some of which amplify inflammation and, consequently, induce CID, were induced by IH and were involved in the proliferation of VSMCs. Since IH increased IL-6 and epiregulin expression in VSMCs, the same phenomenon may also occur in other smooth muscle cells, and, consequently, may be related to the incidence or progression of several diseases. In the present review, we describe how IH can induce the excessive proliferation of VSMCs and we develop the suggestion that other CID may be related to the effects of IH on other smooth muscle cells.

Development of a novel aortic dissection mouse model and evaluation of drug efficacy using in-vivo assays and database analyses.

OBJECTIVE: Aortic dissection is a life-threatening disease. At present, the only therapeutic strategies available are surgery and antihypertensive drugs. Moreover, the molecular mechanisms underlying the onset of aortic dissection are still unclear. We established a novel aortic dissection model in mice using pharmacologically induced endothelial dysfunction. We then used the Japanese Adverse Drug Event Report database to investigate the role of pitavastatin in preventing the onset of aortic dissection. METHODS AND RESULTS: To induce endothelial dysfunction, Nω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, was administered to C57BL/6 mice. Three weeks later, angiotensin II (Ang II) and ß-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, were administered with osmotic mini-pumps. False lumen formation was used as the pathological determinant of aortic dissection. The incidences of aortic dissection and death from aneurysmal rupture were significantly higher in the Nω-nitro-L-arginine methyl ester, Ang II, and BAPN (LAB) group than they were in the Ang II and BAPN (AB) group.Pitavastatin was administered orally to LAB mice. It significantly lowered the incidences of dissection and rupture. It also decreased inflammation and medial degradation, both of which were exacerbated in the LAB group. The Japanese Adverse Drug Event Report database analysis indicated that there were 113 cases of aortic dissection out of 95 090 patients (0.12%) not receiving statins but only six cases out of 16 668 patients receiving statins (0.04%) (odds ratio: 0.30; P = 0.0043). CONCLUSION: Our results suggest that endothelial dysfunction is associated with the onset of aortic dissection and pitavastatin can help prevent this condition.

Nitrosonifedipine, a Photodegradation Product of Nifedipine, Suppresses Pharmacologically Induced Aortic Aneurysm Formation.

BACKGROUND/AIMS: We have reported that nitrosonifedipine (NO-NIF), a photodegradation product of nifedipine, has strong antioxidant and endothelial protective effects, and can suppress several cardiovascular diseases in animal models. The objective of the present study was to investigate the effects of NO-NIF on aortic aneurysm formation. METHODS: The mice were infused with ß-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. The oxidative stress was measured by dihydroethidium staining and nitrotyrosine staining. The expressions of inflammation-related genes were assessed by quantitative real-time PCR and immunohistochemical staining. To clarify the mechanisms of how NO-NIF suppresses vascular cell adhesion molecule (VCAM)-1, endothelial cells were used in in vitro system. RESULTS: NO-NIF suppressed pharmacologically induced the aortic aneurysm formation and aortic expansion without blood pressure changes. NO-NIF suppressed elastin degradation and matrix metalloproteinase-2 mRNA expression. NO-NIF suppressed the reactive oxygen species-cyclophilin A positive feedback loop. Upregulated mRNA expressions of inflammation-related genes and endothelial VCAM-1 were suppressed by NO-NIF co-treatment in aortae. CONCLUSION: NO-NIF has the potential to be a new, nifedipine-derived therapeutic drug for suppressing aortic aneurysm formation by directly improving aortic structure with its strong ability to reduce oxidative stress and inflammation.

Intermittent hypoxia-induced epiregulin expression by IL-6 production in human coronary artery smooth muscle cells.

Patients with obstructive sleep apnea (OSA) experience repetitive episodes of desaturation and resaturation of blood oxygen (known as intermittent hypoxia or IH), during sleep. We showed previously that IH induced excessive proliferation of rat vascular smooth muscle cells through upregulation of members of the epidermal growth factor family, especially epiregulin (EREG), and the erbB2 receptor. In this study, we exposed human coronary artery smooth muscle cells to IH and found that IH significantly increased the expression of EREG. IH increased the production of interleukin-6 (IL-6) in smooth muscle cells, and the addition of IL-6 induced EREG expression. Small interfering RNA for IL-6 or IL-6 receptor attenuated the IH-induced increase in EREG. IL-6 may play a pivotal role in EREG upregulation by IH and consequently OSA-related atherosclerosis.

[Vascular smooth muscle cell response to cyclic mechanical stretch and aortic dissection].

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. The pulsatile nature of blood flow exposes vascular smooth muscle cells (VSMCs) in the vessel wall to cyclic mechanical stretch (CMS), which evokes VSMC death, phenotypic switching, and migration, leading to aortic dissection. We have revealed that CMS of rat aortic smooth muscle cells (RASMCs) caused JNK- and p38-dependent cell death and that a calcium channel blocker, azelnidipine and an angiotensin II receptor antagonist, olmesartan decreased the phosphorylation of JNK and p38 and, subsequently, decreased cell death by CMS. JNK and p38 inhibitors also inhibited CMS-induced cell death. In addition, we showed that the expression of Cxcl1 and Cx3cl1 chemokines was induced by CMS in a JNK-dependent manner. Expression of Cxcl1 was also induced in VSMCs by hypertension produced by abdominal aortic constriction in mouse. In addition, antagonists against the receptors for CXCL1 and CX3CL1 increased cell death, indicating that CXCL1 and CX3CL1 protect RASMCs from CMS-induced cell death. We also revealed that STAT1 is activated in RASMCs subjected to CMS. Taken together, these results indicate that CMS of VSMCs induces inflammation-related gene expression, including that of CXCL1 and CX3CL1, and activates JNK and p38 MAP kinases, which may play important roles in the stress response against CMS caused by acute rise in blood pressure.

Chemokines protect vascular smooth muscle cells from cell death induced by cyclic mechanical stretch.

The pulsatile nature of blood flow exposes vascular smooth muscle cells (VSMCs) in the vessel wall to cyclic mechanical stretch (CMS), which evokes VSMC proliferation, cell death, phenotypic switching, and migration, leading to vascular remodeling. These responses have been observed in many cardiovascular diseases; however, the underlying mechanisms remain unclear. We have revealed that CMS of rat aortic smooth muscle cells (RASMCs) causes JNK- and p38-dependent cell death and that a calcium channel blocker and angiotensin II receptor antagonist decreased the phosphorylation of JNK and p38 and subsequently decreased cell death by CMS. In the present study, we showed that the expression of Cxcl1 and Cx3cl1 was induced by CMS in a JNK-dependent manner. The expression of Cxcl1 was also induced in VSMCs by hypertension produced by abdominal aortic constriction (AAC). In addition, antagonists against the receptors for CXCL1 and CX3CL1 increased cell death, indicating that CXCL1 and CX3CL1 protect RASMCs from CMS-induced cell death. We also revealed that STAT1 is activated in RASMCs subjected to CMS. Taken together, these results indicate that CMS of VSMCs induces inflammation-related gene expression, including that of CXCL1 and CX3CL1, which may play important roles in the stress response against CMS caused by hypertension.

Detection of Nitric Oxide Induced by Angiotensin II Receptor Type 1 Using Soluble Guanylate Cyclase beta1 Subunit Fused to a Yellow Fluorescent Protein, Venus.

Nitric oxide (NO) is an important gaseous molecule involved in many physiological and pathophysiological processes, including the regulation of G protein-coupled receptors (GPCRs). Here, we report the development of a high-affinity method to detect NO using soluble guanylate cyclase beta1 subunit fused to Venus, a variant of yellow fluorescent protein (sGC-Venus). We measured the fluorescence intensity of sGC-Venus with and without an NO donor using purified probes. At 560 nm emission, the fluorescence intensity of sGC-Venus at 405 nm excitation was increased by approximately 2.5-fold by the NO donor, but the fluorescence intensities of sGC-Venus excited by other wavelengths showed much less of an increase or no significant increase. To measure NO in living cells, the fluorescence intensity of sGC-Venus at 405 nm excitation was normalized to that at 488 nm excitation because it showed no significant difference with or without the NO donor. In HEK293 cells overexpressing the angiotensin II receptor type 1 (AT1 receptor), the production of NO induced by activation of the AT1 receptor was detected using sGC-Venus. These data indicate that sGC-Venus will be a useful tool for visualizing intracellular NO in living cells and that NO might be a common tool to regulate GPCRs.

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways.

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.

Olmesartan inhibits cultured rat aortic smooth muscle cell death induced by cyclic mechanical stretch through the inhibition of the c-Jun N-terminal kinase and p38 signaling pathways.

Acute aortic dissection (AAD) is a life-threating disease; however, there is almost no effective pharmacotherapy for it. An increase in c-Jun N-terminal kinase (JNK) phosphorylation and smooth muscle cell (SMC) apoptosis is observed tissues in patients with AAD. Therefore, we hypothesized that an acute rise in blood pressure leads to SMC death through phosphorylation of JNK or p38, which may cause AAD. We investigated the influence of cyclic mechanical stretch, which mimics an acute increase in blood pressure, on cultured rat aortic SMCs (RASMCs) and examined the changes in JNK and p38 phosphorylation. Further, we investigated the effect of olmesartan, an angiotensin II receptor blocker, on stretch-induced RASMC death. We found that mechanical stretch-induced RASMC death in a time-dependent manner, which correlated with the phosphorylation of JNK and p38. Olmesartan inhibited RASMC death and the phosphorylation of JNK and p38. JNK and p38 inhibitors reversed stretch-induced RASMC death. These results suggest that acute mechanical stretch causes JNK and p38 phosphorylation, which may result in SMC death leading to aortic dissection. Olmesartan may be used for pharmacotherapy to prevent aortic dissection, independent of its blood pressure-lowering effect, through its inhibition of JNK and p38 phosphorylation.

Azelnidipine inhibits cultured rat aortic smooth muscle cell death induced by cyclic mechanical stretch.

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor.

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

S-nitrosylation regulates mitochondrial quality control via activation of parkin.

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

Role of big mitogen-activated protein kinase 1 (BMK1) / extracellular signal-regulated kinase 5 (ERK5) in the pathogenesis and progression of atherosclerosis.

Big mitogen-activated protein kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a newly identified member of the mitogen-activated protein (MAP) kinase family. BMK1 has been reported to be sensitive to various neuro-humoral factors and oxidative stress in various cells. In this review, we focused on the role of BMK1 in atherosclerosis in a cultured rat aortic smooth muscle cell model. Treatment with platelet-derived growth factor caused vascular smooth muscle cell (VSMC) migration in a BMK1 activation-dependent manner. H(2)O(2) caused BMK1 activation and VSMC death, including apoptosis of VSMCs. An inhibitory function for BMK1 against cell death from oxidative stress was discovered using siRNA techniques to downregulate the expression of BMK1. These findings suggest a role for BMK1 in the pathogenesis and/or progression of atherosclerosis.