Cell-free DNA testing for early hepatocellular carcinoma surveillance.

BACKGROUND: Liver cirrhosis (LC) is the highest risk factor for hepatocellular carcinoma (HCC) development worldwide. The efficacy of the guideline-recommended surveillance methods for patients with LC remains unpromising. METHODS: A total of 4367 LCs not previously known to have HCC and 510 HCCs from 16 hospitals across 11 provinces of China were recruited in this multi-center, large-scale, cross-sectional study. Participants were divided into Stage Ⅰ cohort (510 HCCs and 2074 LCs) and Stage Ⅱ cohort (2293 LCs) according to their enrollment time and underwent Tri-phasic CT/enhanced MRI, US, AFP, and cell-free DNA (cfDNA). A screening model called PreCar Score was established based on five features of cfDNA using Stage Ⅰ cohort. Surveillance performance of PreCar Score alone or in combination with US/AFP was evaluated in Stage Ⅱ cohort. FINDINGS: PreCar Score showed a significantly higher sensitivity for the detection of early/very early HCC (Barcelona stage A/0) in contrast to US (sensitivity of 51.32% [95% CI: 39.66%-62.84%] at 95.53% [95% CI: 94.62%-96.38%] specificity for PreCar Score; sensitivity of 23.68% [95% CI: 14.99%-35.07%] at 99.37% [95% CI: 98.91%-99.64%] specificity for US) (P < 0.01, Fisher's exact test). PreCar Score plus US further achieved a higher sensitivity of 60.53% at 95.08% specificity for early/very early HCC screening. INTERPRETATION: Our study developed and validated a cfDNA-based screening tool (PreCar Score) for HCC in cohorts at high risk. The combination of PreCar Score and US can serve as a promising and practical strategy for routine HCC care. FUNDING: A full list of funding bodies that contributed to this study can be found in Acknowledgments section.

Identification of thrombotic biomarkers in orthopedic surgery patients by plasma proteomics.

BACKGROUND: Due to the poor specificity of D-dimer, more accurate thrombus biomarkers are clinically needed to improve the diagnostic power of VTE. METHODS: The plasma samples were classified into low-risk group (n = 6) and high-risk group (n = 6) according to the Caprini Thrombosis Risk Assessment Scale score. Data-independent acquisition mass spectrometry (DIA-MS) was performed to identify the proteins in the 12 plasma samples. Bioinformatics analysis including volcano plot, heatmap, KEGG pathways and chord diagram analysis were drawn to analyze the significantly differentially expressed proteins (DEPs) between the two groups. Then, another 26 plasma samples were collected to verify the key proteins as potential biomarkers of VTE in orthopedic surgery patients. RESULTS: A total of 371 proteins were identified by DIA-MS in 12 plasma samples. Volcano plotting showed that there were 30 DEPs. KEGG pathway enrichment analysis revealed that the DEPs were majorly involved in the blood coagulation pathway. The chord diagram analysis demonstrated that proteins SAA1, VWF, FLNA, ACTB, VINC, F13B, F13A and IPSP in the DEPs were significantly related to blood coagulation. VWF and F13B were selected for validation experiments. ELISA test showed that, as compared with those in the low-risk group, the level of VWF in the high-risk sera was significantly increased. CONCLUSIONS: The level of VWF in the high-risk group of thrombosis after orthopedic surgery was significantly higher than that in the low-risk group of preoperative thrombosis, suggesting that VWF may be used as a potential thrombus biomarker in orthopedic surgery patients.

The development of a cSMART-based integrated model for hepatocellular carcinoma diagnosis.

BACKGROUND: Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising. METHODS: Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes. RESULTS: An integrated diagnostic model called "Combined method" was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

Calorie restriction remodels gut microbiota and suppresses tumorigenesis of colorectal cancer in mice.

Colorectal cancer (CRC) is one of the most common cancers worldwide and the consumption of a high-calorie diet is one of its risk factors. Calorie restriction (CR) slows tumor growth in a variety of cancers, including colorectal cancer; however, the mechanism behind this remains unknown. In the present study, CR effectively reduced the tumor volume and weight in a xenograft BALB/c male nude mouse model. In addition, tumor immunohistochemistry revealed that the CR group had significantly higher expression of Bax (P<0.001) and significantly lower levels of Bcl2 (P<0.0001) and Ki67 (P<0.001) compared with control group. Furthermore, data from 16S ribosomal (r)RNA sequencing implied that CR was able to reprogram the microbiota structure, characterized by increased Lactobacillus constituent ratio (P<0.05), with amelioration of microbial dysbiosis caused by CRC. Further receiver operating characteristic curves demonstrated that the bacteria Bacteroides [area under the curve (AUC)=0.800], Lactobacillus (AUC=0.760) and Roseburia (AUC=0.720) served key roles in suppression of CRC in the mouse model. The functional prediction of intestinal flora indicated 'cyanoamino acid metabolism' (P<0.01), 'replication initiation protein REP (rolling circle plasmid replication)' (P<0.01), 'tRNA G10 N-methylase Trm11' (P<0.01) and 'uncharacterized protein with cyclophilin fold, contains DUF369 domain' (P<0.05) were downregulated in CR group. These findings implied that CR suppressed CRC in mice and altered the gut microbiota.

The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients.

PURPOSE: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. EXPERIMENTAL DESIGN: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. RESULTS: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. CONCLUSIONS: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Development of a novel 7 immune-related genes prognostic model for oral cancer: A study based on TCGA database.

Oral squamous cell carcinoma (OSCC) is an aggressive tumor whose prognosis has little improvement in the last three decades. Various immune-related genes have been suggested as significant roles in the development and progression of malignant cancers. In this study, we acquired and integrated differentially expressed genes of OSCC patients, including immune-related genes and transcription factors (TFs), from The Cancer Genome Atlas (TCGA) database. TF-mediated network was established to exploring the regulatory mechanisms of prognostic immune-related genes. A 7 immune-related genes prognostic model for OSCC was obtained, including CGB8, CTLA4, TNFRSF19, CCL26, NRG1, TPM2 and PLAU, which was further proved to be an independent prognostic indicator after adjusting for other clinical factors. The immune-related genes prognostic index was significantly negatively correlated to the infiltration abundances of B cells (P < 0.05) and CD8+ T cells (P < 0.05). The novel proposed immune-based prognostic model not only provided a promising biomarker and a way to monitor the long-term treatment of OSCC, but also gave a new insight into a potential immunotherapy strategy.

A new compound from an endophytic fungus Bambusicola massarinia.

One new chromone, rel-(1S,2S,3S)-2,8-dihydroxy-6-methoxy-1,3-dimethyl-3,4-dihydro-1H-xanthen-9(2H)-one (1), together with one known compound wentiquinone A (2), were isolated from solid culture of endophytic fungus strain Bambusicola massarinia. The structures of all compounds were determined mainly by analysis of their NMR spectroscopic data. The relative configuration of compound 1 was determined by the single-crystal X-ray diffraction analyses.

Mas receptors in modulating relaxation induced by perivascular adipose tissue.

AIMS: Perivascular adipose tissue (PVAT) is known to secrete vascular relaxation factors, and angiotensin 1-7 [Ang-(1-7)] acting on the endothelium is one of the endothelium-dependent relaxation factors. Mas protein is the receptor for Ang-(1-7). Using aorta from Mas-knockout (K/O) and wild type (FVB) mice, we wished to establish the essential role of Mas receptors in mediating the endothelium-dependent relaxation response induced by relaxation factors from PVAT. MAIN METHODS: Thoracic aortic rings from K/O and FVB mice were prepared with or without PVAT (PVAT+ and PVAT-) and/or intact endothelium (E+) or with the endothelium removed (E-) for functional studies. The contraction and relaxation responses of these vessels to agonist in the presence of different receptor antagonists were studied. KEY FINDINGS: PVAT attenuated the contraction induced by phenylephrine (PHE) in the presence of endothelium only in vessels from FVB mice. Mas receptor antagonists D-Ala-Ang-(1-7) (A779) or D-Pro(7)-Ang-(1-7) enhanced the contraction induced by PHE only in vessels from FVB mice. Ang-(1-7) caused a relaxation response only in E+vessels from FVB mice. Transfer of donor solution from PVAT+ vessels to PVAT- recipient vessels caused a relaxation response among FVB vessels and not among vessels from K/O mice. SIGNIFICANCE: Mas receptors are essential in mediating the endothelium-dependent relaxation response induced by PVAT, therefore highlighting the important role of Ang-(1-7) in the control of vascular functions through PVAT.

Alterations in perivascular adipose tissue structure and function in hypertension.

We studied the structural and the functional alterations of perivascular adipose tissue (PVAT) in hypertension with spontaneously hypertensive rats (SHR). Measured with dual energy X-ray absorptiometry, a smaller body fat mass and a greater lean mass were found in SHR than in Wistar-Kyoto (WKY) rats, while body weight was comparable between them. In the thoracic PVAT, the density and the total number of brown adipocytes were greater in SHR than in WKY rats, while the cross section area of PVAT was similar between them. In functional assessment, four types of vessel preparations (with either intact PVAT or intact endothelium, or with both, or without both) were employed. Vessels with intact PVAT from SHR contracted more to phenylephrine than that from WKY rats, while vessels without PVAT exhibited comparable contractile response to phenylephrine between SHR and WKY rats. Both endothelium-dependent and -independent components of PVAT-associated attenuation of phenylephrine-induced contraction were reduced in SHR as compared with that of WKY rats. Bioassay experiments were carried out to assess the transferable relaxation factor from the PVAT. Transfer of bathing solution incubated with PVAT-intact vessel caused less relaxation in SHR recipients than in WKY rats, and the relaxation response was abolished by D-Ala(7)-angiotensin-(1-7), an angiotensin-(1-7) receptor antagonist. In summary, PVAT-associated inhibition of vessel contractile response to agonist was impaired in SHR, and the impairment involved both endothelium-dependent and -independent mechanisms. The functional impairment observed in SHR PVAT may be related to changes in adipocyte composition but not to reduced PVAT mass in SHR.

Modulation of vein function by perivascular adipose tissue.

Although a number of studies have shown that perivascular adipose tissue (PVAT) attenuates arterial contraction through the release of perivascular-derived relaxation factors (PVRF), the role of PVAT in modulating venous function and its mechanism(s) remained unknown. Here we examined the role of PVAT in the modulation of vascular function in the inferior vena cava. Venous rings from male Wistar rats were prepared with both endothelium and PVAT intact, with either PVAT or endothelium removed, or with both endothelium and PVAT removed for functional studies. Contractile response to phenylephrine, U 46619, or 5-hydroxytryptamine was significantly attenuated in PVAT+ as compared with PVAT- veins. PVAT- vessels with intact endothelium (E+) pre-contracted with phenylephrine showed a concentration-dependent relaxation response to angiotensin 1-7 [Ang-(1-7)], and this response was abolished by the removal of endothelium, and by Ang-(1-7) (Mas) receptor antagonists D-Ala-Ang-(1-7) (A779) or D-Pro(7)-Ang-(1-7). Donor solution incubated with a PVAT+ ring induced a relaxation response in the E+ recipient vessel but not in E- recipient vessel. The use of specific channel blockers and enzyme inhibitors showed that Ang-(1-7) is a transferable PVRF that induces endothelium-dependent relaxation through NO release and activation of voltage-dependent potassium (K(+)) channels (K(v)) channels. We conclude that venous PVAT attenuates agonist-induced contraction by releasing Ang-(1-7), which causes relaxation of smooth muscle through endothelial NO release and activation of K(v) channels.

[Screening of breast cancer antigen from a phage display random peptide library].

AIM: to isolate novel breast cancer antigen for developing cancer marker and vaccine, a phage display peptide library was screened with serum antibody from breast cancer patients. METHODS: a phage display peptide library was screened with serum IgG from six breast cancer patients, and then identified the positive clones by ELISA. According to the sequences of the positive clones, the synthesized oligonucleotides of one small peptide (P15) was cloned into PGEX-4T-1. Finally the fusion protein (GST-P15), which was purified from E.coli. BL21, was checked its binding activity with two kinds of serum antibodies (20 breast cancer patients and 20 normal people) by ELISA. RESULTS: 38 positive clones which specifically bound with serum IgG from breast cancer patients were screened. Some of these clones had the same DNA sequence. A small peptide (P15) and purified its fusion protein (GST-P15) was obtained successfully. ELISA assay showed GST-P15 had higher affinity with the serum antibody from breast cancer patients than with the normal serum and there was a significant difference (P<0.01). CONCLUSION: the peptide P15 can be recognized specifically by serum antibody from the breast cancer patients and they will be helpful for isolate new cancer antigen from breast cancer.

Mechanisms for perivascular adipose tissue-mediated potentiation of vascular contraction to perivascular neuronal stimulation: the role of adipocyte-derived angiotensin II.

In rat mesenteric arteries we have recently found that perivascular adipose tissue (PVAT) promoted vasoconstriction to perivascular neuronal activation (by electrical field stimulation, EFS) through generation of superoxide. In this study, we examined the role of adipocyte-generated angiotensin II in PVAT-mediated potentiation of contraction to nerve stimulation. In rat mesenteric PVAT, the presence of angiotesinogen and angiotensin I-converting enzyme (ACE) mRNA was confirmed by RT-PCR. Immunohistochemical staining showed the presence of angiotensin II in mesenteric PVAT. In rat mesenteric arteries, treatment of the vessels with an ACE inhibitor (enalaprilat) or angiotensin II type 1 receptor antagonist (candesartan) reduced PVAT-mediated potentiation of EFS-induced contraction. Exogenously applied angiotensin II enhanced EFS-induced contraction in arteries without PVAT, but not in the arteries with intact PVAT. Chronic treatment with an ACE inhibitor quinapril (14 days) lowered blood pressure and alleviated the potentiation effects of PVAT in EFS-induced contraction. Mesenteric arteries from quinapril-treated group now exhibited the potentiation response to exogenously applied angiotensin II in arteries with intact PVAT to a comparable level as in arteries with PVAT removed. Treatment with hydralazine reduced blood pressure to the same level as quinapril treatment, but did not affect PVAT-associated potentiation of vasoconstriction to EFS and the response to exogenously applied angiotensin II in PVAT-intact arteries. These results showed that adipocyte-derived angiotensin II is critically involved in PVAT-mediated potentiation of EFS-evoked contraction in rat mesenteric arteries.

Alteration of perivascular adipose tissue function in angiotensin II-induced hypertension.

We studied the role of perivascular adipose tissue (PVAT) in the control of vascular function in an in vivo experimental model of hypertension produced by angiotensin II infusion by osmotic minipump in adult male Wistar rats. Two weeks after infusion with angiotensin II, blood pressure in treated rats was significantly elevated but heart rate was reduced compared with control rats infused with physiological saline. Contraction of aorta from the 2 groups of rats in response to phenylephrine or serotonin was significantly attenuated by the presence of PVAT in both the presence and absence of endothelium. This attenuation effect on contraction to phenylephrine was higher, however, in vessels from control rats than in vessels from hypertensive rats in the absence of endothelium. In the mesenteric resistance arteries, lumen diameter was larger in both hypertensive and control vessels with intact PVAT than in vessels with PVAT removed. The medial wall was thicker in arteries from hypertensive rats. The presence of PVAT potentiated the contraction induced by KCl in mesenteric arteries from control rats, but not in hypertensive rats. PVAT also attenuated the contraction of mesenteric arteries in response to phenylephrine or serotonin in both hypertensive and control groups. Mesenteric arteries from hypertensive rats were more responsive to stimulation by serotonin than those from control rats. We conclude that the increased blood pressure of Wistar rats that occurred after infusion with angiotensin II was associated with changes in the functions of PVAT in the aorta and mesenteric arteries and in the structure and function of resistance arteries.

Endothelium-dependent relaxation factor released by perivascular adipose tissue.

OBJECTIVE: Recent studies have demonstrated that perivascular adipose tissue (PVAT) releases vascular relaxation factor(s), but the identity of this relaxation factor remains unknown. Here, we examined if angiotensin 1-7 [Ang-(1-7)] is one of the relaxation factors released by PVAT. METHOD: Morphological and functional methods were used to study aorta from adult Wistar rats. RESULTS: Immunohistochemical staining showed abundant presence of Ang-(1-7) in aortic PVAT. In vessels with PVAT removed but intact endothelium (PVAT - E+), contraction induced by phenylephrine was attenuated by preincubation with Ang-(1-7). PVAT - E+ vessels precontracted with phenylephrine showed a concentration-dependent relaxation response to Ang-(1-7), and this response was abolished by the removal of endothelium. Relaxation response induced by Ang-(1-7) was also prevented by Ang-(1-7) receptor (Mas) antagonist (A779), nitric oxide synthase inhibitor, and nitric oxide scavenger. Ang-(1-7) did not cause a relaxation response in aorta precontracted with KCl, and the relaxation response to Ang-(1-7) was also blocked by calcium-dependent potassium (K(Ca)) channel blockers. Incubation of PVAT + E+ vessels with A779 or angiotensin-converting enzyme 2 inhibitor DX600 or angiotensin-converting enzyme inhibitor enalaprilat increased the contraction induced by phenylephrine. Transfer of donor solution incubated with PVAT + E+ vessel to recipient PVAT - E+ vessel caused a relaxation response. This relaxation response was abolished when donor vessels were incubated with DX600 or enalaprilat or when recipient vessels were incubated with A779. CONCLUSION: Ang-(1-7) released by PVAT acts on the endothelium to cause the release of nitric oxide, and nitric oxide acts as a hyperpolarizing factor through K(Ca) channels to cause relaxation of the blood vessel.

Effects of hyperglycemia on the modulation of vascular function by perivascular adipose tissue.

OBJECTIVE: To study the acute and chronic effect of hyperglycemia on perivascular adipose tissue (PVAT) function in rat aorta. METHOD: Alterations in PVAT function in rat aorta incubated with 22 mmol/l D-glucose for 30 min and in aorta from streptozotocin (STZ)-induced diabetic rats were studied. RESULTS: Incubation with D-glucose caused an attenuation of contraction in response to phenylephrine, both in the presence and absence of endothelium, whereas removal of PVAT eliminated this attenuation effect. The presence of PVAT did not affect concentration-related relaxation response of the aorta to carbamylcholine in STZ rats. There was also no difference in the relaxation response of the aorta to carbamylcholine between STZ and control rats. The presence of PVAT, however, caused a higher attenuation of the concentration-dependent contraction to phenylephrine in aorta from STZ rats with intact endothelium as compared with that from control rats. Incubation of the aorta from control rats with Nomega-nitro-L-arginine or carboxy-2-phenyl-4,4,5,5-tetra-methyl-imidazoline-1-oxyl-3-oxide potentiated the contraction of the vessels to phenylephrine, and this potentiation effect was higher in the vessels from STZ rats than control rats when N-nitro-L-arginine was used. Removal of PVAT reduced this potentiation effect and eliminated the difference between the vessels from control and STZ rats. CONCLUSION: Under both acute and chronic conditions, hyperglycemia enhanced the relaxation response of the vessels mediated by PVAT. These new findings provide important information on the mechanism underlying the postprandial effect of hyperglycemia on blood pressure control and the presence of hypotension under chronic hyperglycemia in a type-1 model of diabetes.

Flow-induced vascular remodeling in the mesenteric artery of spontaneously hypertensive rats.

The effect of an increased blood flow on vascular remodeling was studied in the mesenteric arteries of 11-12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). Increased blood flow was induced by selective ligation of mesenteric arteries. Nearby arteries with normal blood flow were used as controls. 7-10 days after the ligation procedure, mesenteric arteries were fixed in situ at maximal relaxation by perfusion fixation. Morphometric measurement of vascular dimension was carried out with confocal microscopy. Apoptotic cells were detected by the TdT-mediated dUTP nick-end labelling method. Cell growth was quantified by using proliferating cell nuclear antigen (PCNA) in sections of paraffin-embedded vessels. In SHR, elevated blood flow increased the vessel wall dimension and the number of smooth muscle cell (SMC) layers and also increased the wall-to-lumen ratio and the number of PCNA-positive SMC, but did not change lumen size or number of apoptotic SMC. In WKY, on the other hand, increased blood flow resulted in an increase in lumen diameter, a reduction of apoptotic SMC, but no change in wall-to-lumen ratio, number of SMC layers, or number of PCNA-positive SMC. These results showed that mesenteric arteries from hypertensive and normotensive rats respond to an increase in blood flow differently: a lumen enlargement with reduced SMC apoptosis in WKY, but an increased wall-to-lumen ratio with enhanced SMC growth in SHR. Although it remains to be determined whether flow alteration is one of the initiating factors in the development of vascular remodeling in hypertension, we speculate that an increase in cardiac output, and therefore an increase in shear stress that occurs in young SHR, contributes to vascular remodelling in this model of hypertension.

[A case-control study on risk factors for tuberculosis in Yinchuan city].

OBJECTIVE: To study the risk factors of tuberculosis in Yinchuan city and lay a basis for its prevention and control. METHODS: A matched case-control (119:179) study for the risk factors was carried out. Data were analyzed with single-variable analysis and multiple factor logistic regression analysis. RESULTS: Single-variable analysis showed that the education background (chi2 = 2.363, P = 0.018), family economic income (chi2 = 3.040, P = 0.002), smoking (chi2 = 2.500, P = 0.012), physical activities (chi2 = 2.330, P = 0.020), bacille Calmette-Guerin (BCG) vaccination history (chi2 = 22.151, P = 0.000), history of exposure to tuberculosis (chi2 = 15.740, P = 0.000) and so on had significant effects on tuberculosis. Multiple logistic regression analysis showed that family monthly income, smoking, physical activity, BCG vaccination history, history of exposure to tuberculosis entered the final regression model (chi2 = 5.880, 7.368, 3.891, 21.127, 14.536; OR = 0.529, 1.571, 0.774, 0.264, 3.978; P < 0.05). CONCLUSION: History of exposure to tuberculosis and smoking should be the risk factors of tuberculosis in Yinchuan. Having much income, physical activities, and BCG vaccination history should be the preventive factors.

Effects of fetal and neonatal exposure to nicotine on blood pressure and perivascular adipose tissue function in adult life.

In Wistar rats, maternal exposure to nicotine was shown to impair the inhibitory function of perivascular adipose tissue on vascular contractility in the aorta of the offspring. It is not known whether an impairment of perivascular adipose tissue function occurs in smaller arteries, and whether the control of blood pressure is affected. Here we studied the blood pressure effects and the alteration of perivascular adipose tissue function in mesenteric arteries of the offspring born to Wistar-Kyoto rat (WKY) dams exposed to nicotine. Nulliparous female WKY rats were given either nicotine bitartrate (1 mg/kg/day) or saline (vehicle) by subcutaneous injection 2 weeks prior to mating, during pregnancy and until weaning. Blood pressure of the offspring and functional studies with mesenteric arteries were conducted. Tissue samples (thoracic aorta, mesenteric arteries, and kidneys) were collected for morphological and immunohistochemical examinations. Blood pressure increased from 14 weeks of age onwards in the offspring born to nicotine-exposed dams. Nicotine-exposed offspring showed a significant increase in the number of brown adipocytes in aortic perivascular adipose tissue relative to control offspring. In mesenteric arteries from control offspring, contractile responses induced by phenylephrine, serotonin, and 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F(2)alpha (U44619) were significantly attenuated in the presence of perivascular adipose tissue, an effect not observed in the nicotine-exposed tissues. Endothelium-dependent relaxation responses to carbachol, kidney weight, the total number of nephrons and glomerulus' size were comparable in nicotine and saline groups. We conclude that fetal and neonatal exposure to nicotine caused blood pressure elevation. Alterations in perivascular adipose tissue composition and modulatory function are some of the mechanisms associated with this blood pressure increase.

Superoxide anion mediates angiotensin II-induced potentiation of contractile response to sympathetic stimulation.

Angiotensin II is known to potentiate vasoconstriction induced by electrical field stimulation (EFS), but the underlying mechanisms for this potentiation are not fully understood. This study was designed to investigate the role of superoxide anion in the potentiation effects of angiotensin II. Contraction of rat mesenteric arterial segments was induced by perivascular nerve stimulation with EFS, and superoxide production was measured with lucigenin-enhanced chemiluminescence. Extracellular signal-regulated kinase (ERK) phosphorylation was determined in cultured smooth muscle cells with Western blot. Angiotensin II concentration dependently potentiated the contraction of rat mesenteric arteries to EFS, which is frequency-dependent. This potentiation was blunted by an angiotensin AT(1) receptor antagonist (2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid, CV-11974), NAD(P)H oxidase inhibitor (apocynin), superoxide dismutase (SOD) and its mimetic tiron, but not affected by angiotensin AT(2) receptor antagonist and inhibitors of xanthine oxidase, cytochrome P450, and cyclooxygenase. Angiotensin II increased superoxide production by mesenteric arteries, which was blunted by angiotensin AT(1) receptor antagonist CV-11974, and NAD(P)H oxidase inhibitor apocynin. Superoxide generating compound pyrogallol mimicked the effects of angiotensin II. Tyrosine kinase inhibitor (tyrphostin A25) and mitogen-activated protein kinase (MAPK)/ERK inhibitors (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene (U 0126)) inhibited angiotensin II- and pyrogallol-induced potentiation of EFS-induced contraction, while inactive forms of these inhibitors did not show any inhibitory effects. In cultured smooth muscle cells from mesenteric arteries, angiotensin II and superoxide similarly induced ERK phosphorylation. These results showed that superoxide mediated angiotensin II-induced potentiation of contractile response to EFS and tyrosine kinase-MAPK/ERK activation was involved.