HMGB2 Release Promotes Pulmonary Hypertension and Predicts Severity and Mortality of Patients With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which involves aberrant proliferation and apoptosis resistance of the pulmonary arterial smooth muscle cells (PASMCs), resembling the hallmark characteristics of cancer. In cancer, the HMGB2 (high-mobility group box 2) protein promotes the pro-proliferative/antiapoptotic phenotype. However, the function of HMGB2 in PH remains uninvestigated. METHODS: Smooth muscle cell (SMC)-specific HMGB2 knockout or HMGB2-OE (HMGB2 overexpression) mice and HMGB2 silenced rats were used to establish hypoxia+Su5416 (HySu)-induced PH mouse and monocrotaline-induced PH rat models, respectively. The effects of HMGB2 and its underlying mechanisms were subsequently elucidated using RNA-sequencing and cellular and molecular biology analyses. Serum HMGB2 levels were measured in the controls and patients with pulmonary arterial (PA) hypertension. RESULTS: HMGB2 expression was markedly increased in the PAs of patients with PA hypertension and PH rodent models and was predominantly localized in PASMCs. SMC-specific HMGB2 deficiency or silencing attenuated PH development and pulmonary vascular remodeling in hypoxia+Su5416-induced mice and monocrotaline-treated rats. SMC-specific HMGB2 overexpression aggravated hypoxia+Su5416-induced PH. HMGB2 knockdown inhibited PASMC proliferation in vitro in response to PDGF-BB (platelet-derived growth factor-BB). In contrast, HMGB2 protein stimulation caused the hyperproliferation of PASMCs. In addition, HMGB2 promoted PASMC proliferation and the development of PH by RAGE (receptor for advanced glycation end products)/FAK (focal adhesion kinase)-mediated Hippo/YAP (yes-associated protein) signaling suppression. Serum HMGB2 levels were significantly increased in patients with PA hypertension, and they correlated with disease severity, predicting worse survival. CONCLUSIONS: Our findings indicate that targeting HMGB2 might be a novel therapeutic strategy for treating PH. Serum HMGB2 levels could serve as a novel biomarker for diagnosing PA hypertension and determining its prognosis.

Elevated serum levels of leukocyte cell-derived chemotaxin 2 are associated with the prevalence of metabolic syndrome.

AIMS: Inflammation is central to the pathogenesis of metabolic syndrome (MetS). Leukocyte cell-derived chemotaxin 2 (LECT2) is constitutively secreted in response to inflammatory stimuli and oxidative stress contributing to tissue or systemic inflammation. We explored the relationship between LECT2 levels and MetS severity in humans and mice. METHODS: Serum LECT2 levels were measured in 210 participants with MetS and 114 without MetS (non-MetS). LECT2 expression in the liver and adipose tissue was also examined in mice fed a high-fat diet (HFD) and genetically obese (ob/ob) mice. RESULTS: Serum LECT2 levels were significantly higher in MetS participants than in non-MetS participants (7.47[3.36-17.14] vs. 3.74[2.61-5.82], P < 0.001). Particularly, serum LECT2 levels were significantly elevated in participants with hypertension, central obesity, diabetes mellitus (DM), hyperglycaemia, elevated triglyceride (TG) levels, and reduced high-density lipoprotein cholesterol (HDL-C) levels compared to those in participants without these conditions. Pearson's correlation analysis showed that serum LECT2 levels were positively associated with conventional risk factors in all patients. Moreover, LECT2 was positively associated with the number of MetS components (r = 0.355, P < 0.001), indicating that higher serum LECT2 levels reflected MetS severity. Multivariate regression analysis revealed that a one standard deviation increase in LECT2 was associated with an odds ratio of 1.52 (1.01-2.29, P = 0.044) for MetS prevalence after adjusting for age, sex, body mass index, waist circumference, smoking status, white blood cell count, fasting blood glucose, TG, total cholesterol, HDL-C, blood urea nitrogen, and alanine aminotransferase. Receiver operating characteristic curve analysis confirmed the strong predictive ability of serum LECT2 levels for MetS. The optimum serum LECT2 cut-off value was 9.05. The area under the curve was 0.73 (95% confidence interval 0.68-0.78, P < 0.001), with a sensitivity and specificity of 45.71% and 95.61%, respectively. Additionally, LECT2 expression levels were higher at baseline and dramatically enhanced in metabolic organs (e.g. the liver) and adipose tissue in HFD-induced obese mice and ob/ob mice. CONCLUSIONS: Increased LECT2 levels were significantly and independently associated with the presence and severity of MetS, indicating that LECT2 could be used as a novel biomarker and clinical predictor of MetS.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

BACKGROUND: T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS: Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS: LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS: LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Why pendulum symmetry is absent from the cymose partial inflorescences of Cannaceae? Insights into the essential characteristic of cincinni.

In a typical cincinnus, the neighboring two flowers are generally enantiomorphic, which leads to the pendulum symmetry of the entire cyme. While in a two-flowered Cannaceae cincinnus, the flowers develop the same chirality. In this study, we observed several abnormal cincinni of Canna indica that extended longer than their normal form, which presented a second enantiomorphic flower, thus reflecting a typical pendulum symmetry. The chirality change of the second flower was strongly associated with the position of the lateral cincinnus meristem, which determines the angle size of the cincinnus zigzag shift and may serve as a key factor controlling the formation of pendulum symmetry. We propose that alternating floral chirality and the concomitant pendulum symmetry are the essential characteristics of a typical cincinnus. Accordingly, Canna flowers with the same chirality are arranged in modified cincinni.

Legumain is a predictor of all-cause mortality and potential therapeutic target in acute myocardial infarction.

The prognostic impact of extracellular matrix (ECM) modulation and its regulatory mechanism post-acute myocardial infarction (AMI), require further clarification. Herein, we explore the predictive role of legumain-which showed the ability in ECM degradation-in an AMI patient cohort and investigate the underlying mechanisms. A total of 212 AMI patients and 323 healthy controls were enrolled in the study. Moreover, AMI was induced in mice by permanent ligation of the left anterior descending artery and fibroblasts were adopted for mechanism analysis. Based on the cut-off value for the receiver-operating characteristics curve, AMI patients were stratified into low (n = 168) and high (n = 44) plasma legumain concentration (PLG) groups. However, PLG was significantly higher in AMI patients than that in the healthy controls (median 5.9 µg/L [interquartile range: 4.2-9.3 µg/L] vs. median 4.4 µg/L [interquartile range: 3.2-6.1 µg/L], P < 0.001). All-cause mortality was significantly higher in the high PLG group compared to that in the low PLG group (median follow-up period, 39.2 months; 31.8% vs. 12.5%; P = 0.002). Multivariate Cox regression analysis showed that high PLG was associated with increased all-cause mortality after adjusting for clinical confounders (HR = 3.1, 95% confidence interval (CI) = 1.4-7.0, P = 0.005). In accordance with the clinical observations, legumain concentration was also increased in peripheral blood, and infarcted cardiac tissue from experimental AMI mice. Pharmacological blockade of legumain with RR-11a, improved cardiac function, decreased cardiac rupture rate, and attenuated left chamber dilation and wall thinning post-AMI. Hence, plasma legumain concentration is of prognostic value in AMI patients. Moreover, legumain aggravates cardiac remodelling through promoting ECM degradation which occurs, at least partially, via activation of the MMP-2 pathway.

Can the anatomy of abnormal flowers elucidate relationships of the androecial members in the ginger (Zingiberaceae)?

BACKGROUND: Interpretation of the floral structure of Zingiberaceae has long concentrated on the relationships of the androecial members. It suggested that labellum is composed of two structures rather than three or five, and glands are interpreted either as gynoecial part or as androecial members. METHODS: Serial sections were used to observe the vasculature of normal and two-staminate flowers in Alpinia intermedia 'shengzhen'. Floral diagrams were drawn to interpret the morphological category of the floral organs and the relationships of the androecial members. Androecial vascular bundles were associated with carpellary dorsal bundles (CDBs) and parietal bundles (PBs) in a Zingiberales phylogeny setting using ancestral state reconstruction. RESULTS: Anatomical observations demonstrate that the fertile stamen(s) incorporate parietal bundles both in normal and two-staminate flowers. The three appendages represent the three members of the outer whorl of the androecium, while the labellum represents the inner whorl of the androecium in the two-staminate flower. Reconstruction of the origin of the vascular system in the androecium suggests that the outer whorl of androecium receives its vascular supply from the CDBs, and the inner whorl of androecium receives from the PBs in both the basal banana group and the more derived ginger clade. CONCLUSIONS: The present study adds to a growing body of literature suggesting that the anatomy of abnormal flowers may not provide enough evidence for elucidating the relationships of the androecial members, and help us to better understand how the vascular system is constructed during the androecial petaloidy evolution.

Efficacy and Safety of Inorganic Nitrate Versus Placebo Treatment in Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is common, yet there is a lack of effective treatments. In this meta-analysis, we assessed the efficacy and safety of inorganic nitrate in patients with HFpEF. METHODS AND RESULTS: We systematically searched PubMed, Embase, and the Cochrane Library from the inception of the database through March 2020. We included randomized controlled trials that compared the efficacy and safety of inorganic nitrate with a placebo in the treatment of patients with HFpEF. The primary outcome of the meta-analysis was exercise capacity (measured as a change in peak oxygen uptake). We also assessed the effect of inorganic nitrate on diastolic function (measured as changes in E/A and E/e', assessed by echocardiography), quality of life (estimated using the Kansas City Cardiomyopathy Questionnaire), and rest and exercise hemodynamics (measured by invasive cardiac catheterization). In the pooled data analysis, there were no significant differences in peak oxygen uptake (mL/kg/min) [mean difference (MD), 0.25; 95% CI, - 0.07 to 0.57], diastolic function [E/A-standardized mean difference (SMD), 0.51; 95% CI, - 0.17 to 1.20; or E/e'-SMD, 0.02; 95% CI, - 0.23 to 0.27], or quality of life. However, a significant change was observed in the rest and exercise hemodynamics between the inorganic nitrate and placebo treatment in HFpEF patients. No study has reported the effect of inorganic nitrate on hospitalization and mortality of patients with HFpEF. CONCLUSIONS: In patients with HFpEF, the use of inorganic nitrate is not associated with improvements in exercise capacity, diastolic function, and quality of life but is associated with significant changes in rest and exercise hemodynamics.

Osteoprotegerin promotes intimal hyperplasia and contributes to in-stent restenosis: Role of an αVß3/FAK dependent YAP pathway.

OBJECTIVE: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are related to in-stent-restenosis (ISR) following percutaneous coronary intervention (PCI). Osteoprotegerin (OPG) has been implicated in various vascular diseases. However, the effects of OPG on ISR and the underlying mechanism remained elusive. We here investigated the association between OPG and ISR, and to demonstrate the role and potential mechanisms of OPG in neointimal hyperplasia. APPROACH AND RESULTS: From 2962 patients who received coronary angiography and follow-up coronary angiography at approximately one year, 291 patients were diagnosed with ISR, and another 291 gender- and age- matched patients without ISR were selected as controls. Serum OPG levels were significantly increased in patients with ISR. Multivariable logistic regression analysis indicated that OPG level was independently associated with the increased risk of ISR. In a mouse femoral artery wire injury model, upregulated OPG was evidenced in vascular tissue after injury. OPG deletion attenuated the vascular injury-induced neointimal hyperplasia and related gene expression in mice. OPG promoted neointimal hyperplasia and human aortic smooth muscle cell (hASMC) proliferation and migration through activation of yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, whereas knockdown or inhibition of YAP in hASMCs blunted OPG-induced above effects. Moreover, we found that OPG, as a ligand for integrin αVß3, mediated phosphorylation of focal adhesion kinase (FAK) and actin cytoskeleton reorganization, resulting in YAP dephosphorylation in hASMCs. OPG-dependent YAP and VSMC activation was prevented by treatment with αVß3-blocking antibodies and inhibitors of FAK and actin stress fibers. CONCLUSIONS: Increased serum OPG levels are associated with increased risk of ISR following PCI and OPG could promote neointimal hyperplasia in response to injury through integrin αVß3 mediated FAK and YAP activation, indicating OPG/YAP inhibition might serve as an attractive novel target for the prevention of ISR after PCI.

Blood Collection Through Subclavian Vein Puncture in Mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

RNA-Seq analysis reveals the distinctive adaxial-abaxial polarity in the asymmetric one-theca stamen of Canna indica.

Proper establishment of adaxial-abaxial polarity is essential for the development of lateral organs, while former researches were mostly focused on the polarity regulation in leaves, and little is known in stamens, especially in the asymmetric ones. Canna indica (Zingiberales: Cannaceae) is a widely cultivated ornamental plant and the representative species to study the evolutionary development of Zingiberales. The androecium of Canna indica comprises 3-4 petaloid staminodes and a fertile stamen (FS), which consists of a one-theca anther and a petaloid appendage. The partially petaloid stamen is considered as an intermediate state organ from a two-thecae stamen to a completely petaloid staminode. Using RNA-Seq, we quantified the expressions of the transcripts in anther and petaloid appendage, and detected 64,430 and 57,041 unigenes in these two organs, respectively. 4574 unigenes were down-regulated, and 3525 were up-regulated in petaloid appendage compared with those in anther. GO enrichment analysis indicated that the function of cytokinin is more related to cell differentiation in anther, while auxin is more to cell division in petaloid appendage. B- and C-class floral homeotic genes were expressed in these two androecium parts. Most of the class III HD-ZIP family members, which specify adaxial identity, were expressed lower in petaloid appendage than in anther; while KANADIs and YABBYs, which promote abaxial identity, exhibited opposite expression patterns. In situ hybridization showed that the adaxial marker gene was mainly expressed in the region between the two protrusions of the anther, while the abaxial marker was mainly expressed in petaloid appendage. We hypothesize that the adaxial-abaxial polarity participates in the distinctive anther-petaloid appendage patterning within the asymmetric FS of Canna indica.

Molecular basis of floral petaloidy: insights from androecia of Canna indica.

Floral organs that take on the characteristics of petals can occur in all whorls of the monocot order Zingiberales. In Canna indica, the most ornamental or 'petaloid' parts of the flowers are of androecial origin and are considered staminodes. However, the precise nature of these petaloid organs is yet to be determined. In order to gain a better understanding of the genetic basis of androecial identity, a molecular investigation of B- and C-class genes was carried out. Two MADS-box genes GLOBOSA (GLO) and AGAMOUS (AG) were isolated from young inflorescences of C. indica by 3' rapid amplification of cDNA ends polymerase chain reaction (3'-RACE PCR). Sequence characterization and phylogenetic analyses show that CiGLO and CiAG belong to the B- and C-class MADS-box gene family, respectively. CiAG is expressed in petaloid staminodes, the labellum, the fertile stamen and carpels. CiGLO is expressed in petals, petaloid staminodes, the labellum, the fertile stamen and carpels. Expression patterns in mature tissues of CiGLO and CiAG suggest that petaloid staminodes and the labellum are of androecial identity, in agreement with their position within the flower and with described Arabidopsis thaliana expression patterns. Although B- and C-class genes are important components of androecial determination, their expression patterns are not sufficient to explain the distinct morphology observed in staminodes and the fertile stamen in C. indica.

Development and characterization of microsatellite markers for Alpinia oxyphylla (Zingiberaceae).

PREMISE OF THE STUDY: Microsatellite loci were isolated and characterized for population genetic studies of Alpinia oxyphylla (Zingiberaceae), a perennial rhizomatous herbaceous plant often used medicinally in China. • METHODS AND RESULTS: A total of 85 loci were identified using a magnetic bead enrichment method, of which 23 were polymorphic. The level of polymorphism was characterized in 32 individuals from two populations; the number of alleles per locus ranged from 1 to 13; and observed heterozygosity and expected heterozygosity varied from 0 to 1 (mean: 0.6441) and 0 to 0.887 (mean: 0.6241), respectively. • CONCLUSIONS: The polymorphic microsatellite markers generated from this study will be useful for genetic diversity and structure analysis of A. oxyphylla.

Intrasweep phase-sensitive optical coherence tomography for noncontact optical photoacoustic imaging.

We introduce a method to extract the photoacoustic (PA) signal from the phase time evolution of an optical coherence tomography (OCT) swept source spectral sweep. This all-optical detection is achieved in a noncontact fashion directly on the sample surface by using its specular reflection. High-speed measurement and referencing allow for close to shot noise limited phase-sensitive detection. It offers a simple way to perform OCT and PA imaging by sharing the same system components.

Dynamic chirp control and pulse compression for attosecond high-order harmonic emission.

We propose a scheme to compensate dynamically the intrinsic chirp of the attosecond harmonic pulses. By adding a weak second harmonic laser field to the driving laser field, the chirp compensation can be varied from the negative to the positive continuously by simply adjusting the relative time delay between the two-color pulses. Using this technique, the compensation of the negative chirp in harmonic emission is demonstrated experimentally for the first time and the nearly transform-limited attosecond pulse trains are obtained.