Is differential expression of p16INK4a based on the classification of uterine smooth muscle tumors associated with a different prognosis? A meta-analysis.

We conducted a meta-analysis to examine p16INK4a expression in uterine smooth muscle tumors (USMTs). Although the prognostic value of tumor suppressor p16INK4a has been elucidated in a variety of cancers and precancerous lesions, its role in USMTs is not well established. We searched PubMed, Web of Science, and Embase for publication son p16INK4a expression in USMTs. Strict inclusion and exclusion criteria were imposed. Risk ratios (RRs) with 95% confidence intervals (95%CIs) were calculated to assess the strength of association. Publication bias was estimated using funnel plots and the Egger's regression test. Twelve eligible studies comprising 661 patients were included. Compared with leiomyoma (LM), the figures for the strength of association were as follows: LM variants (RR = 1.53, 95%CI = 1.03-2.27, P = 0.036, random effect); leiomyosarcoma (LMS) (RR = 3.20, 95%CI = 1.68-6.12, P < 0.001, random effect); and smooth muscle tumors of uncertain malignant potential (STUMP) (RR = 2.90, 95%CI = 1.17-7.21, P = 0.022, random effect). p16INK4a expression was significantly higher in LMS than in LM variants (RR = 3.74, 95%CI = 1.96-7.13, P < 0.001, random effect) or STUMP (RR = 1.67, 95%CI = 1.26-2.23, P < 0.001, fixed effect). There was a significant correlation between overexpressed p16INK4a and recurrence rates of USMTs (RR = 1.85, 95%CI = 1.11-3.10, P = 0.019, fixed effect). p16INK4a over expression is a potential biomarker for diagnosing problematic USMTs and it might indicate a worse prognosis. However, there is currently insufficient evidence to assess the prognostic value of p16INK4a in USMTs.

[Inventory building of phages against extensively drug-resistant Acinetobacter baumannii isolated from wounds of patients with severe burn and related characteristic analysis].

OBJECTIVE: To build inventory of phages against extensively drug-resistant Acinetobacter Baumannii isolated from wounds of inpatients of burn ICU and analyze related characteristics. METHODS: In 2014 and 2015, 131 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from one hospital in Chongqing. In 2015, 98 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from 6 hospitals in Guangdong province. Above-mentioned 229 strains were collected for conducting experiments as follows: (1) Multilocus sequence typing (MLST) of strains isolated from Chongqing and Guangdong province was analyzed. (2) Sewage co-culture method was applied for isolation of phages with above-mentioned strains and sewage from Chongqing and Guangdong province. Numbers of isolated phages and times of successful isolation and unsuccessful isolation were recorded. (3) The most prevalent subtypes of strains from Chongqing and Guangdong province in 2015 were collected, and their phages respectively underwent cross infection with all strains from Chongqing and those from Guangdong province. The lysis ability of phage was observed when phage underwent cross infection with the same subtype of strain or not the same, and the lytic ratio was calculated. (4) Fluid of phage in one type was randomly selected and equally divided into 3 parts, and its titer was determined by double dilution method. Then each part of phage fluid was subdivided into 3 small parts, which were cultured with LB fluid medium and respectively stored under the condition of -20 ℃, 4 ℃, and room temperature. After being stored for 1 month and 2 months, the titer of phage was determined for evaluating stability of phage. Data were processed with Fisher's exact test, chi-square test, and one-way analysis of variance. RESULTS: (1) The major type of strains from Chongqing in 2014 was ST368 (45%, 31/69), and major types of strains from Chongqing in 2015 were ST75 (26%, 16/62) and ST195 (24%, 15/62), while that from Guangdong province in 2015 was ST977 (46%, 45/98). (2) For strains from Chongqing, isolation effect of phage with sewage of Chongqing (8 times of successful isolation with 9 strains of phages and 1 time of unsuccessful isolation) was better than that with sewage of Guangdong province (1 time of successful isolation with 1 strain of phage and 7 times of unsuccessful isolation). For strains from Guangdong province, isolation effect of phage with sewage of Guangdong province (8 times of successful isolation with 6 strains of phages) was better than that with sewage of Chongqing (7 times of unsuccessful isolation with no phage). These differences were statistically significant (P<0.05 or P<0.01). There was no obvious difference in isolation effect of phage between strains from Chongqing with sewage of Chongqing and strains from Guangdong province with sewage of Guangdong province (P>0.05). (3) The ratios of phages of ST75 and ST977 extensively drug-resistant Acinetobacter Baumannii strains lysing the strains with the same type were respectively 13/16 and 8/9, which were obviously higher than those lysing the strains with different type (respectively 11/115 and 3/53, with χ(2) values respectively 48.23 and 68.46, P values below 0.001). (4) Compared with that before storage, titer of phage under storage condition of -20 ℃, 4 ℃, and room temperature for 1 month decreased by approximately 1 order of magnitude, and that for 2 months decreased by approximately 2 orders of magnitude. After being stored for 1 month and 2 months, there were no statistically significant differences in titer of phage among 3 storage conditions (with F values respectively 1.29 and 1.07, P values above 0.05). CONCLUSIONS: This study has successfully built an inventory covering 229 strains of phages of extensively drug-resistant Acinetobacter Baumannii. MLST of extensively drug-resistant Acinetobacter baumannii varies in different area and different time. Phage can be well isolated using sewage with the same source as that of strain. The lysis ability of phage is closely related to the MLST of strains. Inventory of phages should be built according to regional division. Moreover, phage cultured with LB fluid medium shows good stability without special requirements for storage conditions.

[Therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice].

OBJECTIVE: To study the therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice. METHODS: (1) Sixty BALB/c mice were divided into blank control group, sepsis control group, antibiotics treatment group, phage treatment group, and phage control group according to the random number table, with 12 mice in each group. Mice in blank control group were intraperitoneally (the same injection position below) injected with 1 mL normal saline. Mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL extensively drug-resistant Acinetobacter baumannii (the strain was isolated from the blood of a severely burned patient hospitalized in our unit) in the concentration of 5×10(7) colony-forming unit/mL to reproduce sepsis model. Two hours later, mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL saline, 1 mg/mL imipenem/cilastatin, and 1×10(8) plaque-forming unit (PFU)/mL phages screened based on above-mentioned Acinetobacter baumannii (the same phages below) respectively. Mice in phage control group were injected with 1 mL phages in the titer of 1×10(8) PFU/mL. The injection was performed continuously for 7 days in each living mouse, and the survival situation of mice was observed each day to calculate the survival ratio in one week. (2) Another 60 BALB/c mice were grouped and treated as in experiment (1), and the injection was performed continuously for 5 days in each living mouse. On experiment day 2, 4, and 6, 3 mice from each group were selected (if the number of survived mouse in any group was less than 3 at sample collecting, all the survived mice were selected), and blood was drawn to determine white blood cell count (WBC, with 3 samples at each time point in each group). On experiment day 2, blood was drawn from the mice that had their blood taken earlier for bacterial culture, and lung, liver, kidney, and spleen tissue was collected from the same mice. The tissue samples were added to the LB solid medium after being homogenized and diluted for bacterial culture. The content of bacteria was calculated after the bacterial colony number was counted. Data were processed Wilcoxon rank sum test, one-way analysis of variance, LSD test and Kruskal-Wallis rank sum test. RESULTS: (1) On experiment day 7, there were 12, 8, 10, and 12 mice survived in blank control group, antibiotics treatment group, phage treatment group, and phage control group respectively, while no mouse survived in sepsis control group. Compared with that in sepsis control group, the survival ratio of mice was significantly higher in the other four groups (with Z values from 55.635 to 106.593, P values below 0.05). The survival ratio of mice in phage treatment group was slightly higher than that in antibiotics treatment group, without statistically significant difference (Z=2.797, P>0.05). (2) On experiment day 2, WBC data of mice in blank control group, phage treatment group, and phage control group were close[respectively (5.60±0.94)×10(9)/L, (5.16±0.36)×10(9)/L, and (5.26±1.89)×10(9)/L], all significantly lower than the datum in sepsis control group[(8.64±0.64)×10(9)/L, P<0.05 or P<0.01], and the WBC data in the latter two groups were significantly lower than the datum in antibiotics treatment group[(7.80±1.76)×10(9)/L, with P values below 0.05]. On experiment day 4, WBC data of mice in antibiotics treatment group, phage treatment group, and phage control group were close, all significantly lower than the datum in blank control group (P<0.05 or P<0.01), and WBC data in the above-mentioned four groups were all lower than the datum in sepsis control group (with P values below 0.01). On experiment day 6, there was no statistically significant difference in WBC among blank control group, antibiotics treatment group, phage treatment group, and phage control group (χ(2)=4.128, P>0.05). On experiment day 2, respectively 12, 7, and 2 mice were detected as blood bacterial culture-positive in sepsis control group, antibiotics treatment group, and phage treatment group, while no positive result was detected in the other two groups. Positive ratios of blood bacterial culture of mice in blank control group, phage treatment group, phage control group were significantly lower than the ratio in sepsis control group (with χ(2) values from -30.000 to 30.000, P values below 0.01). Positive ratio of blood bacterial culture of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (with χ(2) values respectively 17.500 and -17.500, P values below 0.05). On experiment day 2, except for the kidney tissue of mice in phage treatment group, the bacteria load in each viscus of mice in blank control group, phage treatment group, and phage control group was significantly lower than that in sepsis control group (with χ(2) values from -9.000 to 9.000, P values below 0.01). The bacteria load in kidney of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (with χ(2) values respectively -7.500 and 7.500, P values below 0.05). CONCLUSIONS: Phages can significantly improve survival ratio, control inflammation response, and effectively clean bacteria in lung, liver, spleen, and kidney in treating extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice.

Removal effect of Cr (VI) by adsorbent made from sewage sludge.

Adsorbent made from sewage sludge is used to remove the Cr (VI) in wastewater. In this paper, the adsorption capacity, adsorption mechanism and regeneration of the adsorbent are studied. A systematic research is carried out to investigate the influence of adsorption capacities in various conditions, such as contact time, pH and initial dosage of adsorbent. The results show that: the equilibrium time is 6 h; the optimal pH is 2.5. The adsorption capacity increases as the initial concentration increase and as the dosage of activated carbon decrease. The data of adsorption capacity are more fit in Langmuir isotherms. The adsorption follows a pseudo-second-order kinetic model perfectly. From this research, the adsorption rate controlling step is film diffusion when the Cr (VI) concentration is low; otherwise, the adsorption rate controlling step is the diffusion among particles. The results of regeneration of saturated adsorbent show that the saturated adsorbent can be perfectly regenerated using alkali treatment. The regeneration ratio of the saturated adsorbent can reach more than 90% with proper concentration of alkali.

Surface properties and adsorption characteristics to methylene blue and iodine of adsorbents from sludge.

Adsorbent materials created from wastewater sludge have unique surface characteristics and could be effective in adsorption applications. In this research, the sludge-adsorbents were generated by pyrolyzing mixtures of sewage sludge and H(2)SO(4). Scanning electron microscope (SEM), thermal analysis, X-ray diffraction (XRD) and X-ray photoelectron spectroscope (XPS) were used to analyze the properties of sludge-adsorbent. XPS results show that the adsorbent surface functional groups with high contents of oxygen-containing groups serve as active sites for the adsorption and affect the surface characteristics; the adsorption mechanism of methylene blue (MB) is mainly Brönsted acid-base reaction between the adsorbent surface and MB; and iodine atoms are bonded to the surface of the adsorbent mainly by dispersive interactions rather than by electrostatic interactions. The results also show that H(2)SO(4) level, pyrolysis temperature and sulfuric acid/sludge weight ratio actually affected the adsorption characteristics. Using the conditions (H(2)SO(4) level of 1-18 M, pyrolysis temperature of 650°C, and weight ratio of 0.8), the adsorption capacities for MB and iodine were 74.7-62.3 mg g(-1) and 169.5-209.3 mg g(-1), respectively.

Differences between karyotypically normal and abnormal human embryonic stem cells.

OBJECTIVES: To compare different biological characteristics of human embryonic stem cells (HESCs) between those with normal and those with abnormal karyotype. MATERIALS AND METHODS: Culture-adapted HESCs (chHES-3) with abnormal karyotype were compared with karyotypically normal cells, with regard to pluripotency and differentiation capacity, ultrastructure, growth characteristics, gene expression profiles and signalling pathways. RESULTS: We found a new abnormal karyotype of HESCs. We observed that chHES-3 cells with normal and abnormal karyotypes shared similarities in expression markers of pluripotency; however, karyotypically abnormal chHES-3 cells had a tendency for differentiation towards ectoderm lineages and were easily maintained in suboptimal culturing conditions. Abnormal chHES-3 cells displayed relatively mature cell organelles compared to normal cells, and karyotypically abnormal chHES-3 cells had increased survival and population growth. Genes related to cell proliferation and apoptosis were up-regulated, but genes associated with genetic instability (p53, Rb, BRCA1) were down-regulated in the karyotypically abnormal cells. CONCLUSION: Karyotypically abnormal chHES-3 cells had a more developed capacity for proliferation, resistance to apoptosis and less genetic stability compared to normal chHES-3 cells and may be an excellent model for studying and characterizing initial stages that determine transition of embryonic stem cells into cancer stem cells.

Effects of filtration temperature, humic acid level and alum dose on cryptosporidium sized particle breakthrough.

Recent Cryptosporidium outbreaks have highlighted concerns about filter efficiency and especially particle breakthrough. Understanding the causes of breakthrough is essential, as the parasite cannot be destroyed by conventional disinfection with chlorine. Particle breakthrough depends on many factors. This research aims to investigate the influence of temperature, humic acid (HA) level and chemical dosing on particle breakthrough in filtration. A series of temperatures were set at 5 degrees C, 15 degrees C and 25 degrees C; humic acid level was 5 mg L(-1). Each was combined with a series of Al doses. A laser particle counter was used to assess the particle breakthrough online. Turbidity, zeta potential, and UV254 absorption were measured before and after filtration. The results showed that particle breakthrough was influenced significantly by temperature, humic acid and dosing. Particle breakthrough occurred earlier at lower temperature, while at higher temperature it was reduced at the same coagulant dose. With coagulants, even at low dose, particle breakthrough was significantly reduced. With HA 5 mg L(-1), particle breakthrough was earlier and the amount was much larger than without HA even at high temperature. There was an optimal dose in filtration and it was well correlated with zeta potential.

Angiotensin II stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries: role of extracellular signal-regulated kinases.

OBJECTIVE: This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells. METHODS: Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059. RESULTS: Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2. CONCLUSIONS: Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.

Altered angiotensin II-induced small artery contraction during the development of hypertension in spontaneously hypertensive rats.

This study assessed whether the angiotensin-II (Ang II)-induced contractile responsiveness of resistance arteries is altered during the development of hypertension in spontaneously hypertensive rats (SHR). Structural parameters and Ang II-stimulated contraction were determined in small mesenteric arteries from 6-week-old (phase of developing hypertension) and 21-week-old SHR (phase of established hypertension), compared with age-matched Wistar-Kyoto rats (WKY). To ascertain whether effects were specific for Ang II, contractile responses to another vasoactive agonist, vasopressin (AVP), were also determined. Systolic blood pressure was measured in conscious rats by the tail-cuff method. Segments of third-order mesenteric arteries (approximately 200 microm in diameter and 2 mm in length) were mounted in a pressurized system with the intraluminal pressure maintained at 45 mm Hg. Blood pressure was significantly increased in SHR (P < .001) and was higher in adult than in young SHR (P < .001). Ang II dose-dependently increased contraction, with responses significantly greater (P < .05) in SHR than in age-matched WKY. SHR, in the early phase of hypertension, exhibited significantly augmented contractile responses (Emax = 70 +/- 5%), compared with SHR with established hypertension (Emax = 33 +/- 5%). These effects were not generalized, as responses to AVP were not significantly different between young and adult SHR. Functional Ang II-elicited alterations were associated with structural modifications: 6-week-old SHR had smaller media to lumen ratio compared with 21-week-old SHR (8.1% +/- 0.17% v 10.6% +/- 0.20%, P < .01). In young SHR vessels the media cross-sectional area was unchanged relative to age-matched WKY rats, suggesting eutrophic remodeling (remodeling index 101.4% v 93.3% young v adult), whereas the cross-sectional area of adult vessels was increased in comparison to WKY rats, suggesting mild hypertrophic remodeling (growth index -1.0% v 15.2%, young v adult). In conclusion, the present study demonstrates that in SHR with early hypertension and slight medial thickening, Ang II-mediated vascular contractile responsiveness is significantly augmented compared with SHR with established hypertension and more severe vascular structural changes. These findings indicate attenuation, as hypertension progresses, of the initially enhanced vascular reactivity to Ang II that is present during the development of hypertension in SHR.

Relationship between small-artery structure and systolic, diastolic and pulse pressure in essential hypertension.

OBJECTIVE: Studies of cerebral arterioles have suggested that pulse pressure may be a more important determinant of small-artery structure than systolic, diastolic or mean blood pressure in stroke-prone spontaneously hypertensive rats and in rats with an arterio-venous shunt. A study of small arteries has suggested that this is not the case in human essential hypertension. We therefore investigated the role of hemodynamic determinants on small-artery structure in hypertensive patients. DESIGN AND METHODS: To determine whether pulse pressure contributes to structural alterations in human essential hypertension, small arteries (lumen < 300 microns) were obtained from gluteal subcutaneous biopsies of 40 normotensive subjects aged 40.7 +/- 1.2 years and 45 untreated essential hypertensive humans aged 46.5 +/- 1.3 years. The relationship between the media: lumen ratio of the small arteries and levels of systolic, diastolic and mean blood pressure and pulse pressure was investigated. RESULTS: The media: lumen ratio (5.33 +/- 0.001%) of small gluteal subcutaneous arteries of normotensive subjects was significantly smaller and the lumen diameter (306 +/- 13 microns) significantly larger than in untreated hypertensive patients (7.42 +/- 0.001% and 244 +/- 9.7 microns respectively, P < 0.001). The media: lumen ratio of both groups examined together correlated with systolic blood pressure (r = 0.45, P < 0.001), diastolic blood pressure (r = 0.56, P < 0.001) and mean arterial pressure (r = 0.55, P < 0.001). The media: lumen ratio of vessels from hypertensive patients correlated with diastolic blood pressure (r = 0.22, P < 0.01) but not with systolic or mean blood pressure. There was no correlation between the media: lumen ratio of small gluteal subcutaneous arteries and pulse pressure in this population of normotensive and hypertensive subjects, examined together or separately. CONCLUSION: These results suggest that in 30- to 65-year-old humans with systolodiastolic essential hypertension, pulse pressure does not appear to be an important determinant of small-artery structure.

Mechanics and composition of human subcutaneous resistance arteries in essential hypertension.

Mechanical properties of arteries are altered in some rat models of hypertension, and this may influence peripheral resistance and blood pressure as well as some of the complications of hypertension. It has usually been assumed that arterial wall stiffness is increased in hypertension, although recent studies suggest that this may not necessarily be the case in large arteries. We determined whether the mechanics of human resistance arteries are altered in hypertension. Subcutaneous resistance arteries (lumen diameter<300 microm) were isolated from hypertensive and normotensive subjects of similar ages (46+/-3 and 43+/-4 years, respectively). Vessels were mounted in a pressurized myograph, deactivated, and exposed to intraluminal pressures ranging from 3 to 140 mm Hg. At each pressure, lumen and media dimensions were measured. Media-to-lumen ratio and media width were greater in hypertensive vessels, reducing wall stress (P<0.01), whereas media cross section was similar in vessels from both groups. Isobaric elastic modulus (which is influenced by vessel geometry and by wall component stiffness) was lower in hypertensive vessels (P<0. 01). Stiffness of wall components (slope of incremental elastic modulus versus stress, which is geometry-independent) was significantly lower in hypertensive vessels (8.2+/-0.7) versus normotensive vessels (11.0+/-1.0, P<0.05), whereas distensibility was unchanged. Electron microscopic analysis of the media of the small arteries showed a greater collagen to elastin ratio (P<0.05) in the media of vessels from hypertensive patients. In conclusion, the stiffness of wall components (slope of elastic modulus versus stress) is not increased but is in fact decreased in subcutaneous resistance arteries from patients with mild essential hypertension. Reduced stiffness of resistance arteries from hypertensive patients does not appear to relate to changes in volume density of extracellular matrix components but may be the result of changes in extracellular matrix architecture or cell-matrix attachment, which remains to be established.

Role of extracellular signal-regulated kinases in angiotensin II-stimulated contraction of smooth muscle cells from human resistance arteries.

BACKGROUND: We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. METHODS AND RESULTS: VSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. CONCLUSIONS: These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.

Endothelin-1 gene expression in blood vessels and kidney of spontaneously hypertensive rats (SHR), L-NAME-treated SHR, and renovascular hypertensive rats.

Spontaneously hypertensive rats (SHR), renovascular hypertensive rats [two-kidney one clip Goldblatt (2-K 1C) and 1-K 1C], and SHR treated with the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), do not respond to endothelin (ET) receptor antagonists with a decrease in blood pressure. However, treatment with ET receptor antagonists has shown some beneficial renal and coronary effects in SHR. In this study we examined tissues from SHR, L-NAME-treated SHR, 2-K 1C, and 1-K 1C, using in situ hybridization with a specific rat preproET-1 cRNA probe to evaluate preproET-1 mRNA abundance in blood vessels, heart, and kidneys. Grain density was similar in SHR and Wistar-Kyoto (WKY) rats in all tissues examined. L-NAME-treated SHR showed increased grain density vs. SHR in endothelium of aorta and of small coronary arteries and in kidney glomeruli, but not in renal or mesenteric arteries. 2-K 1C presented increased grain density in coronary arteries and in glomeruli of the unclipped but not the clipped kidney vs. glomeruli of control rats. 1-K 1C rats exhibited increases in preproET-1 mRNA relative to unilaterally nephrectomized control rats in endothelium of aorta and in mesenteric and coronary arteries, but not in renal arteries or glomeruli. None of the hypertensive models studied showed detectable evidence of myocardial overexpression of preproET-1 mRNA. Therefore, tissue-specific enhancement of ET-1 expression may underlie ET-dependent functional alterations and may explain the beneficial effects of ET receptor antagonists in the coronary circulation or in the kidney in some hypertensive models, but not in SHR.

Abnormal adrenal and vascular responses to vasopressin mediated by a V1-vasopressin receptor in a patient with adrenocorticotropin-independent macronodular adrenal hyperplasia, Cushing's syndrome, and orthostatic hypotension.

The elucidation of gastric inhibitory polypeptide-dependent Cushing's syndrome suggested that ectopic expression or increased responsiveness of other adrenal hormone receptors may underlie ACTH-independent macronodular adrenal hyperplasia (AIMAH) or adrenocortical tumors. We studied a 36-yr-old woman with Cushing's syndrome, AIMAH, and orthostatic hypotension. During upright posture, cortisol and aldosterone were stimulated despite suppression of ACTH and renin. Arginine vasopressin (AVP, 10 U im), under dexamethasone suppression, increased plasma cortisol (3.4-fold), aldosterone (67-fold), and androgens in this patient but not in controls. ACTH 1-24, but not desmopressin acetate, angiotensin II, isoproterenol, or other hormones stimulated steroidogenesis in vivo. Plasma AVP was undetectable initially and increased suboptimally during posture tests after bilateral adrenalectomy. AVP stimulated cortisol production more in dispersed cells from the AIMAH than from a normal adult adrenal (424 vs. 135% at 10 nmol/L). Adrenal V1-AVP receptor presence and mediation of response were shown by RT-PCR and by binding and [Ca+2]i studies. Post adrenalectomy, orthostatic hypotension persisted; a prolonged vasoconstrictive response to AVP was found in vitro in the patient's sc small arteries. We propose that altered adrenal and vascular responses of the V1-AVP receptor-effector pathway underlie this new syndrome.

Effect of chronic ETA-selective endothelin receptor antagonism on blood pressure in experimental and genetic hypertension in rats.

1. Chronic treatment with a combined ETA/ETB endothelin receptor antagonist has been shown to reduce blood pressure in experimental rat models of hypertension in which endothelin-1 gene overexpression occurs in the walls of blood vessels, particularly small, resistance-sized arteries, but not in those genetic or experimental models of hypertension in which there is no overexpression of vascular endothelin-1. Failure of some experimental models of hypertension to respond to treatment with the combined ETA/ETB endothelin antagonist may be due in part to blockade of vasorelaxant endothelial ETB receptors which could in theory reduce the efficacy of endothelin antagonism. 2. In this study the orally active ETA-selective endothelin antagonists A-127722.5 and LU 135252 were used in chronic experiments on deoxycorticosterone acetate (DOCA)-salt hypertensive rats (which overexpress vascular endothelin-1 and respond with blood pressure lowering to combined ETA/ETB endothelin receptor antagonism), on spontaneously hypertensive rats (SHR) (which do not overexpress vascular endothelin-1 and do not respond with blood pressure lowering to the combined ETA/ETB receptor antagonist), and in 1-kidney 1 clip Goldblatt (1-K IC) hypertensive rats (which present mild overexpression of vascular endothelin-1 but do not respond with blood pressure lowering to the combined ETA/ETB receptor antagonist). Additionally, it has been suggested that interruption of the renin-angiotensin system may sensitize responses to endothelin antagonism. Accordingly, SHR were treated with an angiotensin converting enzyme inhibitor, cilazapril, in addition to the ETA receptor antagonist. 3. Blood pressure of DOCA-salt hypertensive rats was lowered by a mean of 24 and of 27 mmHg (P < 0.01) by A-127722.5 after 4 weeks of treatment, when given orally at two different doses (10 and 30 mg kg-1 day-1), and by 18 mmHg by LU 135252 50 mg kg-1 day-1. 4. SHR treated with A-127722.5 for 8 weeks starting at 12 weeks of age exhibited the same progressive rise in blood pressure as untreated SHR. Addition of cilazapril resulted in similar reduction of blood pressure in A-127722.5-treated and untreated SHR. 5. Treatment of 1-K IC hypertensive rats with the dose of LU 135252 which lowered blood pressure in DOCA-salt hypertensive rats did not cause any reduction in blood pressure relative to untreated rats. 6. These results demonstrate that treatment with either dose of the selective ETA receptor antagonists A-127722.5 or LU 135252 caused reductions in blood pressure similar to those obtained for a combined ETA/ETB endothelin antagonist. Blood pressure was lowered only in hypertensive rats known to overexpress vascular endothelin-1 (DOCA-salt hypertensive rats) but not in those which do not (SHR) or only have mild vascular overexpression of endothelin-1 gene (1-K 1C hypertensive rats). Reduction in activity of the renin-angiotensin system in SHR did not sensitize blood pressure to potential hypotensive effects of an ETA-selective receptor antagonist.

Enhanced expression of endothelin-1 gene in resistance arteries in severe human essential hypertension.

BACKGROUND: Endothelins are potent vasoconstrictors, and may also act as mitogens and hypertrophic agents. Expression of a member of this family of peptides, endothelin-1, is enhanced in the endothelium of blood vessels of rats with severe forms of hypertension, even in the absence of elevated plasma endothelin levels. In some of these hypertensive models enhanced endothelin-1 gene expression may contribute to vascular hypertrophy of small arteries and to elevation of blood pressure. OBJECTIVE: To establish whether endothelin-1 may play a role in essential hypertension in humans, in whom plasma levels are known to be usually within normal limits, by examining the expression of the endothelin-1 gene in resistance-size arteries of normotensive subjects, and in humans with mild and severe hypertension. METHODS: Using in-situ hybridization, the abundance of endothelin-1 messenger RNA transcripts was evaluated in small arteries of subcutaneous gluteal fat obtained by biopsy in normotensive and hypertensive patients. RESULTS: Vessels from five normotensive subjects and four untreated mild essential hypertensive patients did not exhibit topographically localized specific labeling with the antisense human endothelin-1 probe. Biopsies from four untreated hypertensive patients with moderate-to-severe blood pressure elevation, in contrast, showed a heavy density of grains on endothelial cells of small arteries of gluteal subcutaneous fat, corresponding to hybridization of the antisense human endothelin-1 complementary RNA probe with endothelin-1 messenger RNA. CONCLUSION: Some patients with moderate-to-severe essential hypertension, similar to some experimental rat models with severe blood pressure elevation, exhibit enhanced endothelial expression of the endothelin-1 gene. This is the first demonstration that overexpression of the endothelin-1 gene may occur in the vascular wall in a small sample of this subset of hypertensive patients. This pathophysiologic phenomenon could play a role in blood pressure elevation and perhaps in the pathogenesis of vascular hypertrophy. Treatment with endothelin receptor antagonists may offer a novel therapy for these moderate-to-severe hypertensive patients.

Structure and function of resistance arteries of hypertensive patients treated with a beta-blocker or a calcium channel antagonist.

OBJECTIVE: To investigate the effects on resistance artery structure and function of monotherapy with the beta-blocker atenolol or the calcium channel antagonist nifedipine in its once a day form or gastrointestinal therapeutic system (GITS). SUBJECTS: Twenty well-controlled essential hypertensive patients matched for age, body mass index, duration and severity of hypertension. Normotensive subjects and untreated hypertensives served as the reference groups. METHODS: Resistance-size small arteries (standardized lumen diameter 247 +/- 8 microns) were dissected from a gluteal subcutaneous biopsy, and studied both on a wire myograph as pressurized vessels. RESULTS: The media width:lumen diameter ratio of arteries was 5.37 +/- 0.09% in normotensive subjects, 5.38 +/- 0.18% in patients treated with nifedipine GITS, 6.81 +/- 0.18% in patients treated with atenolol and 7.08 +/- 0.12% in untreated hypertensives (for each of the latter two groups P < 0.001, versus each of the two former groups). The media stress developed in response to noradrenaline and the endothelium-dependent relaxation induced by acetylcholine were significantly smaller in small arteries from untreated or atenolol-treated patients than they were in those from normotensive subjects or nifedipine GITS-treated patients. CONCLUSION: Hypertensive patients with well-controlled blood pressures under treatment for more than 1 year with the once-a-day calcium channel antagonist nifedipine GITS exhibit normal structure and function of gluteal subcutaneous small arteries, whereas similar patients with blood pressure equally well controlled by the beta-blocker atenolol present thicker small arteries with abnormal endothelium-dependent relaxation and altered contractility. Whether this finding applies also to other vascular beds, and whether it is associated with a better outcome in relation to morbidity and mortality resulting from elevated blood pressure, remain to be established.

Localization of sites of enhanced expression of endothelin-1 in the kidney of DOCA-salt hypertensive rats.

Although the role of endothelin-1, a potent vasoconstrictor peptide, in hypertension remains unclear, there is evidence of its involvement in deoxycorticosterone acetate (DOCA)-salt hypertensive rats, in which enhanced vascular production of endothelin-1 has been documented. The study presented here examined endothelin-1 gene expression in the kidney in DOCA-salt hypertensive rats by in situ hybridization histochemistry. A high specific activity 35S-labeled complementary RNA probe was used. Significant increases in abundance of endothelin-1 mRNA transcripts were found in the endothelium of renal vessels, and in capillary endothelial and mesangial cells of glomeruli of the remaining kidney of DOCA-salt hypertensive rats, in comparison with unilaterally nephrectomized control rats. Enhanced expression of the endothelin-1 gene in the kidney of DOCA-salt hypertensive rats may participate in abnormalities of renal function in this model of hypertension, and thus contribute to the development and maintenance of elevated blood pressure.

Differential effects of vasopressin and endothelin-1 on vascular contractile and calcium responses in pressurized small arteries from spontaneously hypertensive rats.

OBJECTIVE: To investigate the effects of vasopressin and endothelin-1 on the intracellular free calcium concentration ([Ca2+]i) and on contractile responses in endothelium-denuded resistance vessels of prehypertensive (5-week-old) and adult hypertensive (17-week-old) spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats. METHODS: Segments (2 mm long) of third-order branches of small mesenteric arteries were mounted in a perfusion myograph and maintained at 60 mmHg pressure. Endothelium was removed by intraluminal passage of air. The vessel [Ca2+]i was measured by fura-2 fluorescence and contraction was determined using a video imaging system to record lumen diameter. RESULTS: Lumen diameter was significantly smaller in 5-and 17-week-old SHR than it was in age-matched WKY rats (5 week-old SHR versus WKY rats: 178 +/- 4.0 versus 195 +/- 4.3 microns; 17-week-old SHR versus WKY rats: 168 +/- 7.0 versus 230 +/- 3.1 microns). The basal [Ca2+]i was significantly higher in 5- and 17-week-old SHR than it was in age-matched WKY rats. Infusions of vasopressin and endothelin-1 increased [Ca2+]i and contractile responses in a dose-dependent manner in all groups. The vasopressin-induced change in [Ca2+]i was significantly greater in 5- and 17-week-old SHR than in age-matched controls. The sensitivity of [Ca2+]i to vasopressin was increased in adult SHR compared with WKY rats (pD2 9.0 +/- 0.1 in SHR, 8.2 +/- 0.3 in WKY rats). Vasopressin-stimulated contractile responses were increased in adult SHR. The endothelin-1-induced change in [Ca2+]i did not differ between WKY rats and SHR. The contractility of vessels in response to endothelin-1 infusion was similar in age-matched groups. CONCLUSIONS: Endothelin-1-induced changes in [Ca2+]i and contractile responses in small arteries are similar in age-matched WKY rats and SHR, whereas responses to vasopressin are significantly enhanced in SHR compared with WKY rats. Thus [Ca2+]i signalling for vasopressin is more active than is that for endothelin-1. Vasopressin but not endothelin-1 might play a role in the development of hypertension in SHR.

Comparison of effect of endothelin antagonism and angiotensin-converting enzyme inhibition on blood pressure and vascular structure in spontaneously hypertensive rats treated with N omega-nitro-L-arginine methyl ester. Correlation with topography of vascular endothelin-1 gene expression.

Inhibition of nitric oxide synthase by L-arginine analogues such as N omega-nitro-L-arginine methyl ester (L-NAME) in spontaneously hypertensive rats (SHR) is associated with malignant hypertension and enhanced expression of the endothelin-1 gene in some blood vessels. In this study, SHR treated chronically with L-NAME (SHR-L-NAME) were given the angiotensin I-converting enzyme inhibitor cilazapril or the endothelin-A/endothelin-B receptor antagonist bosentan for 3 weeks. Systolic pressure was lowered slightly by cilazapril (213 +/- 2 versus 229 +/- 2 mm Hg in untreated SHR-L-NAME, P < .01) but was not significantly lowered by bosentan (223 +/- 2 mm Hg). Hypertrophy of aorta and small arteries (coronary, renal, mesenteric, and femoral) was decreased by cilazapril treatment and unaffected by bosentan. Expression of the endothelin-1 gene was evaluated in SHR-L-NAME by in situ hybridization histochemistry, which showed that endothelin-1 expression was enhanced in the endothelium of aorta but not in small mesenteric arteries in these rats. The absence of enhancement of endothelin-1 gene expression in small arteries may account for the absence of increased severity of hypertrophy of small vessels in SHR-L-NAME and may be a mechanism whereby L-NAME inhibits cardiovascular growth. These results suggest that in the absence of enhanced small-artery endothelin-1 expression, endothelin antagonism does not lower blood pressure. The blood pressure-lowering effect of angiotensin I-converting enzyme inhibition suggests a role for the renin-angiotensin system in the malignant form of hypertension that develops in SHR treated with L-NAME.