A pressor dose of angiotensin II has no influence on the angiotensin-converting enzyme 2 and other molecules associated with SARS-CoV-2 infection in mice.

In the early phase of the Coronavirus disease 2019 (COVID-19) pandemic, it was postulated that the renin-angiotensin-system inhibitors (RASi) increase the infection risk. This was primarily based on numerous reports, which stated that the RASi could increase the organ Angiotensin-converting enzyme 2 (ACE2), the receptor of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in rodents. RASi can theoretically antagonize the potential influence of angiotensin II (Ang II) on ACE2. However, while Ang II decreases the ACE2 levels in cultured cells, there is little evidence that supports this phenomenon in living animals. In this study, we tested whether Ang II or Ang II combined with its antagonist would alter the ACE2 and other molecules associated with the infection of SARS-CoV-2. Male C57BL6/J mice were administered vehicle, Ang II (400 ng/kg/min), or Ang II with losartan (10 mg/kg/min) for 2 weeks. ACE2 knockout mice were used as a negative control for the ACE2 assay. We found that both Ang II, which elevated blood pressure by 30 mm Hg, and Ang II with losartan, had no effect on the expression or protein activity of ACE2 in the lung, left ventricle, kidney, and ileum. Likewise, these interventions had no effect on the expression of Transmembrane Protease Serine 2 (TMPRSS2) and Furin, proteases that facilitate the virus-cell fusion, and the expression or activity of Tumor Necrosis Factor α-Convertase (TACE) that cleaves cell-surface ACE2. Collectively, physiological concentrations of Ang II do not modulate the molecules associated with SARS-CoV-2 infection. These results support the recent observational studies suggesting that the use of RASi is not a risk factor for COVID-19.

A lung abscess caused by secondary syphilis - the utility of polymerase chain reaction techniques in transbronchial biopsy: a case report.

BACKGROUND: In Japan and other countries, the number of patients with syphilis is increasing year by year. Recently, the cases of the pulmonary involvement in patients with secondary syphilis have been reported. However, it is still undetermined how to obtain a desirable specimen for a diagnosis of the pulmonary involvement, and how to treat it if not cured. CASE PRESENTATION: A 34-year-old man presented with cough and swelling of the right inguinal nodes. A physical examination revealed erythematous papular rash over the palms, soles and abdomen. A 4 cm mass in the right lower lobe of the lung was detected on computed tomography. He was diagnosed as having secondary syphilis, because he was tested positive for the rapid plasma reagin and Treponema pallidum hemagglutination assay. Amoxycillin and probenecid were orally administered for 2 weeks. Subsequently, rash and serological markers were improved, however, the lung mass remained unchanged in size. Transbronchial biopsy (TBB) confirmed the pulmonary involvement of syphilis using polymerase chain reaction techniques (tpp47- and polA-PCR). Furthermore, following surgical resection revealed the lung mass to be an abscess. CONCLUSIONS: To our knowledge, this is the first surgically treated case of a lung abscess caused by syphilis, which was diagnosed by PCR techniques in TBB. This report could propose a useful diagnostic method for the pulmonary involvement of syphilis.

Clinical impact of extended blood culture examination: Too much of a good thing.

Blood culture is the most critical examination for diagnosing bacterial infections. The longer the blood culture incubation period, the higher the chances of identifying bacterial strains. However, unnecessary extension of the incubation period can burden the capacity of the instrument and merely result in the detection of contaminant bacteria having no clinical significance. This study aimed to optimize the blood culture incubation period using the currently available continuous-monitoring automated blood culture instrument. This was a 2-year retrospective study performed at Osaka University Hospital (January 1, 2016 to December 31, 2017). The BD BACTEC™ FX blood culture system (Becton Dickinson, Sparks, MD, USA) and BD BACTEC™ Plus series blood culture bottles were used. All blood cultures were incubated for more than 12 consecutive days. We reviewed the clinical data of cases that tested positive between 6 and 12 days of incubation. During the study period, 14,822 sets of blood culture were drawn. Of 1751 sets testing positive, 95.7% (1665 sets) became positive within 5 days of incubation. The overall contamination rate (false positives) after 6 days of incubation was 80.2% (69/86 sets). Based on the positive blood culture results, antimicrobials were changed in 7.0% (6/86) of the sets, and a diagnosis of infectious disease was made in only one case. There was no death associated with the extended blood culture results. In conclusion, the clinical impact of extended blood culture incubation for 6 days or more was limited, and a routine extension of the incubation period might be unnecessary.

Helicobacter cinaedi-associated Carotid Arteritis.

A 65-year-old Japanese man with bilateral carotid atherosclerosis presented with right neck pain and fever. Contrast-enhanced computed tomography suggested carotid arteritis, and carotid ultrasonography showed an unstable plaque. The patient developed a cerebral embolism, causing a transient ischemic attack. Helicobacter cinaedi was detected in blood culture, and H. cinaedi-associated carotid arteritis was diagnosed. Empirical antibiotic therapy was administered for 6 weeks. After readmission for recurrent fever, he was treated another 8 weeks. Although the relationship between H. cinaedi infection and atherosclerosis development remains unclear, the atherosclerotic changes in our patient's carotid artery might have been attributable to H. cinaedi infection.

Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus.

RATIONALE: Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date. OBJECTIVE: The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus. METHODS AND RESULTS: Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter. CONCLUSIONS: In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

Therapeutic vaccine against DPP4 improves glucose metabolism in mice.

The increasing prevalence of type 2 diabetes mellitus is associated with a significant economic burden. We developed a dipeptidyl peptidase 4 (DPP4)-targeted immune therapy to increase glucagon-like peptide 1 hormone levels and improve insulin sensitivity for the prevention and treatment of type 2 diabetes mellitus. Immunization with the DPP4 vaccine in C57BL/6J mice successfully increased DPP4 titer, inhibited plasma DPP4 activity, and induced an increase in the plasma glucagon-like peptide 1 level. Moreover, this elevated titer was sustained for 3 mo. In mice fed a high-fat diet, DPP4 vaccination resulted in improved postprandial glucose excursions and insulin sensitivity and, in the diabetic KK-A(y) and db/db mice strains, DPP4 vaccination significantly reduced glucose excursions and increased both plasma insulin and pancreatic insulin content. Importantly, T cells were not activated following challenge with DPP4 itself, which suggests that this vaccine does not induce cell-mediated autoimmunity. Additionally, no significant immune-mediated damage was detected in cells and tissues where DPP4 is expressed. Thus, this DPP4 vaccine may provide a therapeutic alternative for patients with diabetes.

Segmental aortic stiffening contributes to experimental abdominal aortic aneurysm development.

BACKGROUND: Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined. METHODS AND RESULTS: Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening. CONCLUSIONS: The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.

Cross-talk of receptor activator of nuclear factor-κB ligand signaling with renin-angiotensin system in vascular calcification.

OBJECTIVE: Vascular calcification is accelerated by hypertension and also contributes to hypertension; however, it is an enigma why hypertension and vascular calcification are a vicious spiral. The present study elucidates the cross-talk between renin-angiotensin II system and receptor activator of nuclear factor-κB ligand (RANKL) system in vascular calcification. APPROACH AND RESULTS: Angiotensin (Ang) II (10(-7) mol/L) significantly increased calcium deposition as assessed by Alizarin Red staining, associated with a significant increase in the expression of RANKL, RANK, and bone-related genes, such as cbfa1 and msx2, in human aortic vascular smooth muscle cells. Infusion of Ang II (100 ng/kg per minute) in ovariectomized ApoE(-/-) mice under high-fat diet significantly increased the expression of RANKL system and calcification in vivo, whereas administration of Ang II receptor blocker (olmesartan, 3 mg/kg per day) decreased the calcification and bone markers' expression. In addition, male OPG(-/-) mice showed a significant increase in vascular calcification followed by Ang II infusion as compared with wild type. Conversely, RANKL significantly increased Ang II type 1 receptor and angiotensin II-converting enzyme expression in vascular smooth muscle cells via extracellular signal-regulated protein kinase phosphorylation. CONCLUSIONS: The present study demonstrated that Ang II significantly induced vascular calcification in vitro and in vivo through RANKL activation. In addition, RANKL activated renin-angiotensin II system, especially angiotensin II-converting enzyme and Ang II type 1 receptor. Cross-talk between renin-angiotensin II system and RANKL system might work as a vicious cycle to promote vascular calcification in atherosclerosis. Further studies to inhibit renin-angiotensin II system and RANKL may provide new therapeutic options to prevent and regress vascular calcification.

Micromanaging abdominal aortic aneurysms.

The contribution of abdominal aortic aneurysm (AAA) disease to human morbidity and mortality has increased in the aging, industrialized world. In response, extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of the diseased aorta. This work aims to develop novel diagnostic and therapeutic strategies to limit AAA expansion and, ultimately, rupture. Contributions from multiple research groups have uncovered a complex transcriptional and post-transcriptional regulatory milieu, which is believed to be essential for maintaining aortic vascular homeostasis. Recently, novel small noncoding RNAs, called microRNAs, have been identified as important transcriptional and post-transcriptional inhibitors of gene expression. MicroRNAs are thought to "fine tune" the translational output of their target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. With the discovery that microRNAs act as powerful regulators in the context of a wide variety of diseases, it is only logical that microRNAs be thoroughly explored as potential therapeutic entities. This current review summarizes interesting findings regarding the intriguing roles and benefits of microRNA expression modulation during AAA initiation and propagation. These studies utilize disease-relevant murine models, as well as human tissue from patients undergoing surgical aortic aneurysm repair. Furthermore, we critically examine future therapeutic strategies with regard to their clinical and translational feasibility.

Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification.

RATIONALE: Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor kappaB), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. OBJECTIVE: To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. METHODS AND RESULTS: RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor alpha. Apolipoprotein E-deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. CONCLUSIONS: RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner.

Phase I Study to Assess the Safety and Immunogenicity of an Intradermal COVID-19 DNA Vaccine Administered Using a Pyro-Drive Jet Injector in Healthy Adults.

We conducted a nonrandomized, open-label phase I study to assess the safety and immunogenicity of an intradermal coronavirus disease 2019 (COVID-19) DNA vaccine (AG0302-COVID-19) administered using a pyro-drive jet injector at Osaka University Hospital between Yanagida November 2020 and December 2021. Twenty healthy volunteers, male or female, were enrolled in the low-dose (0.2 mg) or high-dose (0.4 mg) groups and administered AG0302-COVID19 twice at a 2-week interval. There were no adverse events that led to discontinuation of the study drug vaccination schedule. A serious adverse event (disc protrusion) was reported in one patient in the high-dose group, but the individual recovered, and the adverse event was not causally related to the study drug. In the analysis of the humoral immune response, the geometric mean titer (GMT) of serum anti-SARS-CoV-2 spike glycoprotein-specific antibody was low in both the low-dose and high-dose groups (246.2 (95% CI 176.2 to 344.1, 348.2 (95% CI 181.3 to 668.9)) at the 8 weeks after first vaccination. Regarding the analysis of the cellular immune, the number of IFN-γ-producing cells responsive to the SARS-CoV-2 spike glycoprotein increased with individual differences after the first dose and was sustained for several months. Overall, no notable safety issues were observed with the intradermal inoculations of AG0302-COVID19. Regarding immunogenicity, a cellular immune response was observed in some subjects after AG0302-COVID19 intradermal inoculation, but no significant antibody production was observed.

Double Deletion of Angiotensin II Type 2 and Mas Receptors Accelerates Aging-Related Muscle Weakness in Male Mice.

Background The activation of AT2 (angiotensin II type 2 receptor ) and Mas receptor by angiotensin II and angiotensin-(1-7), respectively, is the primary process that counteracts activation of the canonical renin-angiotensin system (RAS). Although inhibition of canonical RAS could delay the progression of physiological aging, we recently reported that deletion of Mas had no impact on the aging process in mice. Here, we used male mice with a deletion of only AT2 or a double deletion of AT2 and Mas to clarify whether these receptors contribute to the aging process in a complementary manner, primarily by focusing on aging-related muscle weakness. Methods and Results Serial changes in grip strength of these mice up to 24 months of age showed that AT2/Mas knockout mice, but not AT2 knockout mice, had significantly weaker grip strength than wild-type mice from the age of 18 months. AT2/Mas knockout mice exhibited larger sizes, but smaller numbers and increased frequency of central nucleation (a marker of aged muscle) of single skeletal muscle fibers than AT2 knockout mice. Canonical RAS-associated genes, inflammation-associated genes, and senescence-associated genes were highly expressed in skeletal muscles of AT2/Mas knockout mice. Muscle angiotensin II content increased in AT2/Mas knockout mice. Conclusions Double deletion of AT2 and Mas in mice exaggerated aging-associated muscle weakness, accompanied by signatures of activated RAS, inflammation, and aging in skeletal muscles. Because aging-associated phenotypes were absent in single deletions of the receptors, AT2 and Mas could complement each other in preventing local activation of RAS during aging.

Disseminated gonococcal infection in a Japanese man with complement 7 deficiency with compound heterozygous variants: A case report.

RATIONALE: Complement deficiency are known to be predisposed to disseminated gonococcal infection (DGI). We herein present a case of DGI involving a Japanese man who latently had a complement 7 deficiency with compound heterozygous variants. PATIENT CONCERNS: A previously healthy 51-year-old Japanese man complained of sudden-onset high fever. Physical examination revealed various skin lesions including red papules on his trunk and extremities, an impetigo-like pustule on left forearm, and tendinitis of his right forefinger. DIAGNOSIS: Blood culture testing detected gram-negative cocci, which was confirmed to be Neisseria gonorrhoeae based on mass spectrometry and a pathogen-specific PCR test. INTERVENTIONS: Screening tests for underlying immunocompromised factors uncovered that complement activities (CH50) was undetectable. With a suspicion of a congenital complement deficiency, genetic analysis revealed rare single nucleotide variants in complement 7 (C7), including c.281-1G>T and a novel variant c.1454C>T (p.A485V). CH50 was normally recovered by adding purified human C7 to the patient's serum, supporting that the patient has C7 deficiency with compound heterozygous variants. OUTCOMES: Under a diagnosis of DGI, the patient underwent an antibiotic treatment with cefotaxime for a week and was discharged without any sequela. LESSONS: DGI is a rare sexually-transmitted infection that potentially induces systemic complications. Complement immunity usually defeats N. gonorrhoeae and prevents the organism from causing DGI. This case highlighted the importance of suspecting a complement deficiency when a person develops DGI.

RAGE ligands stimulate angiotensin II type I receptor (AT1) via RAGE/AT1 complex on the cell membrane.

The receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein α-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-κB activation, and epithelial-mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases.

The endocytosis of oxidized LDL via the activation of the angiotensin II type 1 receptor.

Arrestin-dependent activation of a G-protein-coupled receptor (GPCR) triggers endocytotic internalization of the receptor complex. We analyzed the interaction between the pattern recognition receptor (PRR) lectin-like oxidized low-density lipoprotein (oxLDL) receptor (LOX-1) and the GPCR angiotensin II type 1 receptor (AT1) to report a hitherto unidentified mechanism whereby internalization of the GPCR mediates cellular endocytosis of the PRR ligand. Using genetically modified Chinese hamster ovary cells, we found that oxLDL activates Gαi but not the Gαq pathway of AT1 in the presence of LOX-1. Endocytosis of the oxLDL-LOX-1 complex through the AT1-ß-arrestin pathway was demonstrated by real-time imaging of the membrane dynamics of LOX-1 and visualization of endocytosis of oxLDL. Finally, this endocytotic pathway involving GPCR kinases (GRKs), ß-arrestin, and clathrin is relevant in accumulating oxLDL in human vascular endothelial cells. Together, our findings indicate that oxLDL activates selective G proteins and ß-arrestin-dependent internalization of AT1, whereby the oxLDL-LOX-1 complex undergoes endocytosis.

Pustules on the back possibly triggering toxic-shock syndrome.

We herein describe an irregular case of toxic-shock syndrome (TSS). A previously healthy 28-year-old Japanese man developed a sudden-onset high fever. The patient was suffering from conjunctival hyperaemia, gastrointestinal symptoms such as vomiting and diarrhoea, and systemically diffused macular erythroderma. Further physical examination detected pustules on his back, which self-destructed over time. Laboratory revealed multiple organ failures. Subsequently, scalded skin on the face and desquamation in the limb extremities emerged by day 10, leading to the diagnosis of TSS, despite his stable circulatory dynamics through the course. Learning points for clinicians include that they should recall TSS as a possible disease concurrently causing high fever, systemic rash and multiple organ dysfunctions, even without being in a state of shock. The characteristic desquamations emerged in the limb extremities after hospitalisation were of help in diagnosing TSS.

A case of primary aldosteronism with resistant hypertension successfully treated by unilateral adrenalectomy after unsuccessful classification of subtype in adrenal venous sampling.

Despite being an established method to identify the unilateral subtype of primary aldosteronism with an indication of adrenalectomy, adrenal venous sampling sometimes fails primarily due to unsuccessful cannulation to adrenal veins. In such cases, the analysis of clinical findings might help to identify the indication of surgery.