CYCLOIDEA-like genes control floral symmetry, floral orientation, and nectar guide patterning.

Actinomorphic flowers usually orient vertically (relative to the horizon) and possess symmetric nectar guides, while zygomorphic flowers often face horizontally and have asymmetric nectar guides, indicating that floral symmetry, floral orientation, and nectar guide patterning are correlated. The origin of floral zygomorphy is dependent on the dorsoventrally asymmetric expression of CYCLOIDEA (CYC)-like genes. However, how horizontal orientation and asymmetric nectar guides are achieved remains poorly understood. Here, we selected Chirita pumila (Gesneriaceae) as a model plant to explore the molecular bases for these traits. By analyzing gene expression patterns, protein-DNA and protein-protein interactions, and encoded protein functions, we identified multiple roles and functional divergence of 2 CYC-like genes, i.e. CpCYC1 and CpCYC2, in controlling floral symmetry, floral orientation, and nectar guide patterning. CpCYC1 positively regulates its own expression, whereas CpCYC2 does not regulate itself. In addition, CpCYC2 upregulates CpCYC1, while CpCYC1 downregulates CpCYC2. This asymmetric auto-regulation and cross-regulation mechanism might explain the high expression levels of only 1 of these genes. We show that CpCYC1 and CpCYC2 determine asymmetric nectar guide formation, likely by directly repressing the flavonoid synthesis-related gene CpF3'5'H. We further suggest that CYC-like genes play multiple conserved roles in Gesneriaceae. These findings shed light on the repeated origins of zygomorphic flowers in angiosperms.

Photoluminescent Properties and Mechanism of Novel Cyanine-Borondifluoride Curcuminoid Hybrids as Red-to-Near Infrared and Endoplasmic Reticulum-Targeting Dyes.

Synthesis-oriented design led us to the discovery of a series of novel cyanine-borondifluoride curcuminoid hybrids called Nanchang Red (NCR) dyes that overcome the intrinsic low synthetic yields of symmetrical cyanine-difluoroboronate (BF2 )-hybridized NIR dyes. The hybridization endows NCR dyes with high molar extinction coefficients, efficient red-to-NIR emission, and enlarged Stokes shifts. Quantum chemical calculations revealed that the asymmetrical layout of the three key electron-withdrawing and electron-donating fragments results in a special pattern of partial charge separation and inconsistent degrees of charge delocalization on their π-conjugated backbones. While the nature of the hemicyanine fragment exerts significant influence on the excitation modes of NCR dyes, the borondifluoride hemicurcuminoid fragment is the major contributor to the enlarged Stokes shifts. Cell imaging experiments illustrated that a subtle change in the N-heterocycle of the hemicyanine fragment has a remarkable effect on the subcellular localization of NCR dyes. Unlike other previously reported cyanine-BF2 hybridized dyes, which mainly target mitochondria, the benzothiazole and indole-based NCR dyes accumulate in both the endoplasmic reticulum (ER) and lipid droplets of HeLa cells, whereas the benzoxazole and quinoline-based NCR dyes stain the ER specifically.

Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE-/- mice.

Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced pro-inflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.

The Role of Psychological Stress on Heart Autophagy in Mice With Heart Failure.

OBJECTIVE: Psychological stress in chronic heart failure (CHF) is associated with systemic neurohormonal and immune system responses and increased mortality. Autophagy refers to the biological process of degradation and recycling of dysfunctional cellular components. We investigated the role of psychological stress on autophagy function in CHF mice. METHODS: C57BL/6 mice underwent transverse aortic constriction, with or without combined acoustic and restraint stress, and cardiac function was assessed by echocardiography analysis. Serum corticosterone and angiotensin II (Ang II) were determined using enzyme-linked immunosorbent assay (ELISA). Autophagy and oxidative stress were measured with immunohistochemistry and quantitative polymerase chain reaction, and chloroquine and rapamycin were used to detect autophagy flux. In vivo, cardiomyocytes were cultured with or without Ang II or N-acetylcysteine, and autophagy and oxidative stress were also detected. RESULTS: A 1-week stress exposure significantly increased serum levels of corticosterone and Ang II (p = .000), increased levels of oxidative stress, induced overt heart failure, and increased mortality (p = .002). Furthermore, stress exposure unregulated messenger RNA expression of Bcl-2-interacting coiled-coil protein 1 (10.891 [3.029] versus 4.754 [1.713], p = .001), cysteine-rich domain containing beclin-1 interacting (6.403 [1.813] versus 3.653 [0.441], p = .006), and autophagy 7 (111.696 [4.049] versus 6.189 [1.931], p = .017), increased expression of autophagosomal, and decreased clearance of autophagosomes. In vitro, Ang II significantly increased autophagy flux in cultured cardiomyocytes, which could be partly inhibited by N-acetylcysteine. CONCLUSIONS: Psychological stress may contribute to the development of CHF by enhancing heart oxidative stress and impairing autophagy flux.

Chronic psychological stress induces vascular inflammation in rabbits.

Psychological stress is associated with a systemic inflammatory response. It is unclear, however, whether psychological stress contributes to vascular inflammation. Here, we examined the effects of unpredictable chronic mild stress (UCMS) on vascular inflammation in rabbits. One hundred rabbits were randomly divided into control and stress groups. UCMS was induced by a set of defined adverse conditions applied in a shuffled order for 4, 8, 12, or 16 weeks, and rabbits were killed 24 h after the end of the UCMS protocol. Expression of different inflammatory molecules was analyzed by real-time polymerase chain reaction, immunohistochemistry, or enzyme-linked immunosorbent assay. UCMS resulted in depression-like behaviors, decreased body weight gain, and hypertension with no significant effects on serum lipids. Aortic mRNA and protein expression for tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor, and expression of intercellular adhesion molecule-1 (ICAM-1) protein were increased. UCMS increased circulating concentrations of corticosterone, TNF-α, and CRP throughout. Moreover, stress downregulated the expression of endothelial nitric oxide synthase. At 16 weeks of UCMS, macrophage infiltration and lipid accumulation in the subendothelial space were detected in the aorta. In cultured murine vascular smooth muscle cells, treatment with serum from stressed rabbits significantly increased phosphorylation of p38 and c-Jun N-terminal kinase (JNK), and upregulated expression of MCP-1 and ICAM-1 mRNAs, in which the effect was blunted by a TNF-α neutralizing antibody or p38 and JNK inhibitors. Our results indicate that chronic psychological stress induces vascular inflammation via TNF-α and p38/JNK pathways, which may contribute to the development of atherosclerosis.

Unpredictable chronic mild stress promotes atherosclerosis in high cholesterol-fed rabbits.

OBJECTIVES: Chronic psychological stress is associated with an increased risk of atherosclerosis in humans. Experimental studies using various stress models have yielded controversial results. This study investigated the effects of unpredictable chronic mild stress (UCMS) on atherogenesis in New Zealand white rabbits. METHODS: Rabbits were fed with a cholesterol-enriched (1%) diet for 4 to 16 weeks, with or without concomitant UCMS treatment. Atherosclerosis was assessed in the abdominal aorta by serial sectioning and morphological analysis. Expressions of inflammatory factors were measured with immunohistochemistry and quantitative polymerase chain reaction. Serum nitrate/nitrite levels were determined with Griess assay, and corticosterone and inflammatory markers were determined using enzyme-linked immunosorbent assay. RESULTS: High-cholesterol feeding resulted in hypercholesterolemia and formation of atherosclerotic plaques in the aorta. UCMS exposure significantly increased the plaque size (p = .003) and decreased the plaque stability (decreased the contents of collagen and smooth muscle and increased the amount of macrophage and matrix metalloproteinases). The proatherogenic effects of UCMS were unrelated to changes in serum cholesterol level but accompanied by increased blood pressure (p < .001) and vascular inflammation (up-regulation of tumor necrosis factor α, C-reactive protein, and monocyte chemoattractant protein 1, all p values < .01). Serum concentrations of nitrate/nitrite were lower in UCMS-treated animals (p = .01). Vessels from UCMS-treated animals exhibited augmented phosphorylation of p38 and c-Jun N-terminal kinase and activation of nuclear factor κB. CONCLUSIONS: Chronic psychological stress may contribute to the development of atherosclerosis by enhancing vascular inflammation and decreasing endothelial nitric oxide bioavailability.

Loss of myeloid Tsc2 predisposes to angiotensin II-induced aortic aneurysm formation in mice.

RATIONALE: Genetic studies have proved the involvement of Tuberous sclerosis complex subunit 2 (Tsc2) in aortic aneurysm. However, the exact role of macrophage Tsc2 in the vascular system remains unclear. Here, we examined the potential function of macrophage Tsc2 in the development of aortic remodeling and aortic aneurysms. METHODS AND RESULTS: Conditional gene knockout strategy combined with histology and whole-transcriptomic analysis showed that Tsc2 deficiency in macrophages aggravated the progression of aortic aneurysms along with an upregulation of proinflammatory cytokines and matrix metallopeptidase-9 in the angiotensin II-induced mouse model. G protein-coupled receptor 68 (Gpr68), a proton-sensing receptor for detecting the extracellular acidic pH, was identified as the most up-regulated gene in Tsc2 deficient macrophages compared with control macrophages. Additionally, Tsc2 deficient macrophages displayed higher glycolysis and glycolytic inhibitor 2-deoxy-D-glucose treatment partially attenuated the level of Gpr68. We further demonstrated an Tsc2-Gpr68-CREB network in macrophages that regulates the inflammatory response, proteolytic degradation and vascular homeostasis. Gpr68 inhibition largely abrogated the progression of aortic aneurysms caused by Tsc2 deficiency in macrophages. CONCLUSIONS: The findings reveal that Tsc2 deficiency in macrophages contributes to aortic aneurysm formation, at least in part, by upregulating Gpr68 expression, which subsequently drives proinflammatory processes and matrix metallopeptidase activation. The data also provide a novel therapeutic strategy to limit the progression of the aneurysm resulting from Tsc2 mutations.

Severity of Nonalcoholic Fatty Liver Disease is Associated With Cardiovascular Outcomes in Patients With Prehypertension or Hypertension: A Community-Based Cohort Study.

Background: It is unclear whether more severe non-alcoholic fatty liver disease (NAFLD) combined with prehypertension or hypertension is associated with a higher risk of cardiovascular events (CVEs). To evaluate the relationship between the severity of NAFLD and CVEs among patients with prehypertension or hypertension. Methods: In this prospective community-based Kailuan cohort, participants without cardiovascular disease and alcohol abuse, or other liver diseases were enrolled. NAFLD was diagnosed by abdominal ultrasonography. Prehypertension was defined as systolic blood pressure (BP) of 120-139 mmHg or diastolic BP of 80-89 mmHg. Participants with NAFLD were divided into mild, moderate, and severe subgroups. Follow-up for CVEs including myocardial infarction, hemorrhagic stroke, and ischemic stroke. The Cox proportional hazards model was used to estimate hazard ratios and 95% CIs of CVEs according to the severity of NAFLD and hypertensive statutes. The C-statistic was used to evaluate the efficiency of models. Results: A total of 71926 participants (mean [SD] age, 51.83 [12.72] years, 53794 [74.79%] men, and 18132 [25.21%] women) were enrolled in this study, 6,045 CVEs occurred during a median of 13.02 (0.65) years of follow-up. Compared with participants without NAFLD, the hazard ratios of CVEs for patients with mild, moderate, and severe NAFLD were 1.143 (95% CI 1.071-1.221, P < 0.001), 1.218 (95% CI 1.071-1.221, P < 0.001), and 1.367 (95% CI 1.172-1.595, P < 0.001), respectively. Moreover, participants with prehypertension plus moderate/severe NAFLD and those with hypertension plus moderate/severe NAFLD had 1.558-fold (95% CI 1.293-1.877, P < 0.001) and 2.357-fold (95% CI 2.063-2.691, P < 0.001) higher risks of CVEs, respectively, compared with those with normal BP and no NAFLD. Adding a combination of NAFLD and BP status to the crude Cox model increased the C-statistic by 0.0130 (0.0115-0.0158, P < 0.001). Conclusions: Our findings indicated that the increased cardiovascular risk with elevated BP is largely driven by the coexistence of moderate/severe NAFLD, suggesting that the severity of NAFLD may help further stratify patients with prehypertension and hypertension.

Danshensu protects vascular endothelia in a rat model of hyperhomocysteinemia.

AIM: To examine whether danshensu could protect vascular endothelia in a rat model of hyperhomocysteinemia. METHODS: The model was established by feeding rats with a methionine-rich diet (1 g·kg⁻¹·d⁻¹) for 3 months. Immediately following the discontinuation of methionine-rich diet, rats were treated with danshensu (67.5 mg·kg⁻¹·d⁻¹, po) or saline for 3 additional months. One group of rats receiving vitamin mixture (folic acid, vitamin B12 and vitamin B6) was included as a positive control. One group of rats not exposed to methionine-rich diet was also included as a blank control. The expression of tumor necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecule-1 (ICAM-1) protein in the descending aorta was examined using immunohistochemistry and Western blot. Homocysteine and blood concentration of endothelin and nitric oxide (NO) was also examined. RESULTS: Methionine-rich diet resulted in accumulation of "foam cells", up-regulated expression of TNF-alpha and ICAM-1 in the descending aorta, and significantly increased serum homocysteine. Plasma endothelin concentration was significantly increased; NO was decreased. Danshensu treatment, either simultaneous to methionine-rich diet or afterwards, attenuated the above mentioned changes. CONCLUSION: Chronic treatment with danshensu could prevent/attenuate the formation of atherosclerosis. Potential mechanisms include inhibited expression of representative proinflammatory cytokines and adhesion molecules in arterial endothelia. Changes in homocysteine and circulating molecules that control vascular contraction/relaxation via endothelial cells (eg, endothelin and NO) were also implicated.

Effects of Quyu Xiaoban capsules on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris.

OBJECTIVES: To investigate the effects of the Traditional Chinese Medicine (TCM) Quyu Xiaoban capsules (QYXB) on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris (UAP) and phlegm and blood stasis syndrome. DESIGN: Ninety (90) UAP patients were randomly divided into two groups: the control group received a loading dose of 300 mg aspirin and a maintenance dose of 100 mg of aspirin plus baseline therapy for 4 weeks, and the trial group received the same doses of aspirin and baseline therapy plus QYXB for 4 weeks. The severity of anginal attacks, alterations of TCM symptoms and signs, and electrocardiographic (ECG) changes were observed in all patients before and after treatment. Plasma platelet aggregation (PAG) rate and P-selectin level were measured in all patients at baseline and at the end of the fourth week. RESULTS: After treatment for 4 weeks, both group of patients showed improvement in the severity of angina pectoris and TCM symptoms and signs, and there was a significant difference of the total effective rate in clinical improvement between the two groups, whereas no difference of the total effective rate in ECG improvement between the two groups was found. Compared with the baseline level, PAG rate in both groups decreased significantly at the end of the fourth week (63.74 +/- 11.18% versus 55.69 +/- 10.40 % in the control group, and 63.83 +/- 12.70% versus 50.04 +/- 8.91% in the trial group). Similar changes of P-selectin levels were observed in both groups (9.40 +/- 1.25 ng/mL versus 8.90 +/- 1.34 ng/mL in the control group and 9.56 +/- 1.16 ng/mL versus 7.80 +/- 0.98 ng/mL in the trial group). However, both PAG rate and P-selectin level decreased to a greater extent in the trial group than in the control group after treatment, and the difference between treatment was significant (both p<0.05). Nevertheless, these biochemical changes were too small to explain fully the beneficial clinical outcomes achieved by QYXB capsules. CONCLUSIONS: On the background of baseline and aspirin therapy, QYXB capsules significantly attenuated anginal attacks and improved TCM symptoms and signs in patients with UAP, and the exact mechanisms underlying these therapeutic effects remain to be explored.

Effects of Quyu xiaoban capsules on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris.

OBJECTIVES: To investigate the effects of the Traditional Chinese Medicine (TCM) Quyu Xiaoban capsules (QYXB) on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris (UAP) and phlegm and blood stasis syndrome. DESIGN: Ninety patients with UAP were randomly divided into two groups: a control group that received a loading dose of 300 mg aspirin and a maintenance dose of 100 mg of aspirin plus baseline therapy for 4 weeks, and a trial group that received the same doses of aspirin and baseline therapy plus QYXB for 4 weeks. The severity of anginal attacks, alterations of TCM symptoms and signs, and electrocardiographic (ECG) changes were assessed in all patients before and after treatment. The plasma platelet aggregation (PAG) rate and P-selectin level were measured in all patients at baseline and at the end of the fourth week of treatment. RESULTS: After treatment for 4 weeks, both group of patients showed improvement in the severity of angina pectoris and TCM symptoms and signs, but there was a significant difference in the two groups' rates of clinical improvement, whereas the rate of ECG improvement of the two groups showed no difference. As compared with the baseline value, the PAG rate in both groups decreased significantly at the end of the fourth week (63.74 +/- 11.18% vs. 55.69 +/- 10.40% in the control group, and 63.83 +/- 12.70% vs. 50.04 +/- 8.91% in the trial group). Similar changes in P-selectin levels were observed in the two groups (9.40 +/- 1.25 ng/mL vs. 8.90 +/- 1.34 ng/mL in the control group, and 9.56 +/- 1.16 ng/mL vs. 7.80 +/- 0.98 ng/mL in the trial group). However, both the PAG rate and P-selectin level decreased to a greater extent in the trial group than in the control group after treatment, and the difference between the two groups was significant (both p < 0.05). Nevertheless, these biochemical changes were too small to fully explain the beneficial clinical outcomes achieved with QYXB capsules. CONCLUSIONS: In comparison with both the respective baseline values and with aspirin therapy, QYXB capsules significantly attenuated anginal attacks and improved TCM symptoms and signs in patients with UAP. The exact mechanisms underlying these therapeutic effects remain to be explored.

Tongxinluo Protects against Hypertensive Kidney Injury in Spontaneously-Hypertensive Rats by Inhibiting Oxidative Stress and Activating Forkhead Box O1 Signaling.

Hypertension is an independent risk factor for the progression of chronic renal failure, and oxidative stress plays a critical role in hypertensive renal damage. Forkbox O1(FoxO1) signaling protects cells against oxidative stress and may be a useful target for treating oxidative stress-induced hypertension. Tongxinluo is a traditional Chinese medicine with cardioprotective and renoprotective functions. Therefore, this study aimed to determine the effects of Tongxinluo in hypertensive renal damage in spontaneously hypertensive rats(SHRs)and elucidate the possible involvement of oxidative stress and FoxO1 signaling in its molecular mechanisms. SHRs treated with Tongxinluo for 12 weeks showed a reduction in systolic blood pressure. In addition to increasing creatinine clearance, Tongxinluo decreased urinary albumin excretion, oxidative stress injury markers including malondialdehyde and protein carbonyls, and expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and its activity in SHR kidneys. While decreasing phosphorylation of FoxO1, Tongxinluo also inhibited the phosphorylation of extracellular signal-regulated kinase1/2 and p38 and enhanced manganese superoxide dismutase and catalase activities in SHR kidneys. Furthermore, histology revealed attenuation of glomerulosclerosis and renal podocyte injury, while Tongxinluo decreased the expression of α-smooth muscle actin, extracellular matrixprotein, transforming growth factor ß1 and small mothers against decapentaplegic homolog 3,and improved tubulointerstitial fibrosis in SHR kidneys. Finally, Tongxinluo inhibited inflammatory cell infiltration as well as expression of tumor necrosis factor-α and interleukin-6. In conclusion, Tongxinluo protected SHRs against hypertension-induced renal injury by exerting antioxidant, antifibrotic, and anti-inflammatory activities. Moreover, the underlying mechanisms of these effects may involve inhibition of oxidative stress and functional activation of FoxO1 signaling.

Period2 deficiency blunts hypoxia-induced mobilization and function of endothelial progenitor cells.

BACKGROUND: In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice. METHODS AND RESULTS: MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2-/- mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2-/- mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2-/- mice after MI. In vitro, per2-/- EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2-/- mice and inhibited PI3K/Akt expression in per2-/- EPCs under hypoxia. CONCLUSIONS: Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.