Deficiency of cyclin-dependent kinase inhibitors p21 and p27 accelerates atherogenesis in apolipoprotein E-deficient mice

Cyclin-dependent kinase inhibitors, p21^Cip1 and p27^Kip1, are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21^Cip1 or p27^Kip1 in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE^−/− aortae, both apoE^−/−/p21^−/− and apoE^−/−/p27^−/− aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27^Kip1 accelerated plaque formation significantly more than p21^−/− in apoE^−/− mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21^Cip1 and p27^Kip1 accelerates atherogenesis in apoE^−/− mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.     

The role of extracellular calcium in pregnancy-induced attenuation of phenylephrine contraction in rat aorta with functional endothelium

The effect of pregnancy on the supply of calcium ions for the contractile responses of rat aortic rings to phenylephrine was investigated. The contractility of intact aortic rings from pregnant rats, compared with that of similar rings from non-pregnant rats, to phenylephrine and potassium chloride was significantly decreased. Contractions of rings from non-pregnant rats, pretreated with phenylephrine or potassium chloride, in response to calcium chloride were greater than those of similarly treated rings from pregnant rats. When the concentration of calcium chloride in the medium bathing the rings was reduced to 0.8 mmol·l^-1, the contractile response to phenylephrine was significantly (P<0.005) inhibited in rings from both pregnant and non-pregnant rats but to a greater extent in rings from non-pregnant rats. Contractions of aortic rings from pregnant rats in response to phenylephrine in calcium-free medium were similar to those of rings from non-pregnant rats, suggesting equal dependence on calcium from intracellular stores. The results suggest that pregnancy decreased the response to calcium influx into the aortic smooth muscle cells through both receptor-and voltage-operated calcium entry pathways. Since de-endothelialisation reversed the pregnancy-induced diminished contraction to phenylephrine, it is likely that pregnancy interferred with contractions induced by activation of receptors with phenylephrine through enhanced production of endothelium-derived relaxing factor(s).Abbreviations EC₅₀ concentration of drug producing 50% contraction - EDCF endothelium-derived contraction factor - EGTA ethyleneglycol-bis (-aminoethyl ether)-NN tetraacetic acid - PSS physiological salt solution - VSM vascular smooth muscle     

Identification of a novel gene, HCR2, in aorta of hypercoagulable rats using suppression subtractive hybridization

Screening and identification of novel genes involved in hypercoagulable state (HCS) is important in understanding the underlying mechanisms of development of atheroslerosis, which implicated in critical vascular diseases such as coronary heart disease and stroke. HCS was induced in rats with high carbohydrate diet. Subtractive hybridization experiments between aorta tissues from hypercoagulable rats and controls showed that a novel cDNA (GenBank Accession No. AY234417), designated as hypercoagulability related gene-2 (HCR2), was highly expressed in aorta tissue. The predicted protein encoded by HCR2 contains 78 amino acids, which has a theoretical isoelectric point (pI) of 8.59 and molecular weight (MW) of 8841.7 based on sequence analysis. Our data suggest that HCR2 may be involved in HCS and identification of the gene may contribute to our understanding of the mechanisms for the production of a hypercoagulable state of several critical clinical disorders.     

A radioautographic study of the transport of 125I-labeled serum lipoproteins in rat aorta

Summary The transport of ¹²⁵I-labeled serum lipoproteins through the aortic endothelium was studied by radioautography. Rat aorta and heart was perfused in vitro with a medium containing human very low density (VLDL), low density (LDL), high density lipoprotein (HDL), delipidated HDL apolipoprotein or rat HDL. In all lipoproteins more than 95% of the radioactivity was TCA precipitable and lipid radioactivity was from 2–4% in HDL, 4–6% in LDL, 7–15% in VLDL. After 18–60 min of perfusion and wash with unlabeled medium, most of the aortic radioactivity was TCA precipitable and the percent of lipid counts was similar to that in the original lipoprotein. Following perfusion with VLDL, LDL, or HDL the radioautographic reaction was seen over the endothelium, the subendothelial space and the inner media, and was separated by an unlabeled zone from the reaction present over the adventitia. Uniform labeling of the entire wall was found after perfusion with HDL apolipoprotein. The presence of silver grains over endothelial cells in regions rich in plasmalemmal vesicles suggested that these organelles participate in the transport of the labeled lipoprotein, as was shown for lactoperoxidase (Stein and Stein, 1972). The present data indicate that cholesterol may enter the aortic wall as a constituent of lipoprotein particles. Since an HDL particle carries less than 1/20 of the cholesterol present in a LDL particle, it seems that the lower susceptibility of the female to atheromatosis might be related to the higher ratio of HDL to LDL particles in the female serum.The excellent technical help of Miss R. Ben-Moshe, Mrs. A. Mandeles, Mr. G. Hollander and Mrs. Y. Dabach is gratefully acknowledged. This study was supported in part by grants from National Institute of Health (No. 06-101-1), United States Public Health Service; Delegation Generale a la Recherche Scientifique et Technique of the French Government and from the Ministry of Health, the Government of Israel.     

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16^INK4a, p21^WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.     

Research and application of biotechnology in textile industries in China

Textile industry is a conventional and pillar industry in China, which possesses a considerable proportion of the national economy. In recent years, special attention has been paid to the application of biotechnology in textile industries in China. As an interdiscipline between natural science and engineering science, textile biotechnology has much effect on China's textile industry. This paper summarizes current developments and highlights those areas where biotechnology might play an increasingly important role in China's textile industry as follows:

(1) Development of new types of textile fibers and polymers, such as Bt cotton naturally colored cotton, colored silk and silk gene-sequence, spider silk non-wovens, chitin fiber and chitosan derivatives, etc.

(2) Application of enzyme technology in textile wet processing, such as alkaline pectinase, PVA-degrading enzyme, cutinase and catalase used for cotton preparation, neutral cellulase for denim washing, transglutaminase for wool modification, protease for silk degumming as well as pectinase and hemicellulases for retting of bast fibers.

(3) Treatment of textile effluents with biotechnology.

Combined treatment with DPP-4 inhibitor linagliptin and SGLT2 inhibitor empagliflozin attenuates neointima formation after vascular injury in diabetic mice

Incretin therapy has emerged as one of the most popular medications for type 2 diabetes. We have previously reported that the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin attenuates neointima formation after vascular injury in non-diabetic mice. In the present study, we examined whether combined treatment with linagliptin and the sodium glucose cotransporter 2 (SGLT2) inhibitor empagliflozin attenuates neointima formation in diabetic mice after vascular injury. Diabetic db/db mice were treated with 3 mg/kg/day linagliptin and/or 30 mg/kg/day empagliflozin from 5 to 10 weeks of age. Body weight was significantly decreased by empagliflozin and the combined treatment. Blood glucose levels and glucose tolerance test results were significantly improved by empagliflozin and the combined treatment, but not by linagliptin. An insulin tolerance test suggested that linagliptin and empagliflozin did not improve insulin sensitivity. In a model of guidewire-induced femoral artery injury in diabetic mice, neointima formation was significantly decreased in mice subjected to combined treatment. In an in vitro assay using rat aortic smooth muscle cells (RASMC), 100, 500, or 1000 nM empagliflozin significantly decreased the RASMC number in a dose-dependent manner. A further significant reduction in RASMC proliferation was observed after combined treatment with 10 nM linagliptin and 100 nM empagliflozin. These data suggest that combined treatment with the DPP-4 inhibitor linagliptin and SGLT2 inhibitor empagliflozin attenuates neointima formation after vascular injury in diabetic mice in vivo and smooth muscle cell proliferation in vitro.     

The pet dogs ability for learning from a human demonstrator in a detour task is independent from the breed and age

There are many indications and much practical knowledge about the different tasks which various breeds of dogs are selected for. Correspondingly these different breeds are known to possess different physical and mental abilities. We hypothesized that commonly kept breeds will show differences in their problem solving ability in a detour task around a V-shaped fence, and also, that breed differences will affect their learning ability from a human demonstrator, who demonstrates a detour around the fence. Subjects were recruited in Hungarian pet dog schools. We compared the results of the 10 most common breeds in our sample when they were tested in the detour task without human demonstration. There was no significant difference between the latencies of detour, however, there was a trend that German Shepherd dogs were the quickest and Giant Schnauzers were the slowest in this test. For testing the social learning ability of dogs we formed three breed groups (“utility”, “shepherd” and “hunting”). There were no significant differences between these, all the breed groups learned equally well from the human demonstrator. However, we found that dogs belonging to the “shepherd” group looked back more frequently to their owner than the dogs in the “hunting” group. Further, we have found that the age of pet dogs did not affect their social learning ability in the detour task. Our results showed that the pet status of a dog has probably a stronger effect on its cognitive performance and human related behaviour than its age or breed. These results emphasize that socialization and common activities with the dog might overcome the possible breed differences, if we give the dogs common problem solving, or social learning tasks.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

Comparative proteomic analysis of rat aorta in a subtotal nephrectomy model

Although accelerated atherosclerosis and arteriosclerosis are the main causes of cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients, the molecular pathogenesis remains largely obscure. Our study of the aortic function in a typical CKD model of subtotal nephrectomy (SNX) rats demonstrated phenotypes that resemble CKD patients with aortic stiffness. The 2‐DE analysis of rat aortas followed by MS identified 29 up‐regulated and 53 down‐regulated proteins in SNX rats. Further Western blot and immunohistochemistry analyses validated the decreased HSP27 and increased milk fat globule epidermal growth factor‐8 (MFG‐E8) in SNX rats. Functional classification of differential protein profiles using KOGnitor revealed that the two major categories involved in aortic stiffness are posttranslational modification, protein turnover, chaperones (23%) and cytoskeleton (21%). Ingenuity Pathway Analysis highlighted cellular assembly and organization, and cardiovascular system development and function as the two most relevant pathways. Among the identified proteins, the clinical significance of the secreted protein MFG‐E8 was confirmed in 50 CKD patients, showing that increased serum MFG‐E8 level is positively related to aortic stiffness and renal function impairment. Drug interventions with an inhibitor of the angiotensin converting enzyme, enalapril, in SNX rats improved aortic stiffness and decreased MFG‐E8 depositions. Together, our studies provide a repertoire of potential biomarkers related to the aortic stiffness in CKD.     

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.     

Hyperlipidemia is a major determinant of neointimal formation in LDL receptor-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice exhibit mild hyperlipidemia on a chow diet but develop severe hyperlipidemia on a high fat diet. In this study, we investigated neointimal formation after removal of the endothelium when LDLR(-/-) mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 10 weeks of age, female mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, neointimal formation was barely detectable in the injured vessel when mice developed mild hyperlipidemia on the chow diet. In contrast, neointimal lesions were obvious when mice developed severe hyperlipidemia on the Western diet. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells and smooth muscle cells in neointimal lesions. The injured artery also exhibited a significant increase in medial area on the Western diet. Plasma levels of MCP-1 and soluble VCAM-1 were significantly elevated by feeding of the Western diet. These data indicate that hyperlipidemia aggravates neointimal growth in LDLR(-/-) mice by promoting foam cell formation and inflammation.     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Age-related change of mineral content in the human thoracic aorta and in the human cerebral artery

The relative contents (RCs) of mineral elements in aortae and cerebral arteries from 23 subjects, with ages ranging between 45 and 99 yr, were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of calcium, phosphorus, and magnesium in the aortae increased markedly after the age of 70. While the RC of sulfur in aortae decreased gradually after that age. It was found that accumulation of calcium and phosphorus occurred primarily in the tunica media of aorta, and secondarily in the tunica intima. Furthermore, the RCs of calcium, phosphorus, and magnesium in cerebral arteries increased markedly after the age of 70, whereas the RC of sulfur in cerebral arteries decreased after age 70. It was found that accumulation of calcium and phosphorus in the cerebral arteries were 30 and 60%, respectively, lower than those in the aortae with ages ranging between 45 and 99 yr.     

Hypoxia-induced increase of endostatin in murine aorta and lung

In the lung, hypoxia induces pulmonary hypertension caused by vasoconstriction and vascular remodeling. Additionally, hypoxia is an inducer of angiogenesis, which is assumed to counteract pulmonary hypertension. We asked whether the anti-angiogenic factor endostatin—a cleavage product of collagen XVIII—participates in the vascular alterations induced by hypoxia. By employing Western blotting of tissue extracts of murine brain, liver and heart an endostatin fragment of 22 kDa was detectable, whereas in lung and aorta additional bands of 24 and 26 kDa were found. The amount of these larger fragments was increased in tissues obtained from mice housed for 4 days or 3 weeks at hypobaric hypoxia. By immunohistochemistry endostatin was detected in association with elastic fibers and in close neighborhood to smooth muscle cells of intrapulmonary vessels and the aorta. In the lung, the activity of matrix metalloproteinases (MMP) known to generate endostatin by cleavage of collagen XVIII was increased (MMP-2) and decreased (proMMP-9), respectively, by hypoxia. Elevated amounts of endostatin within the aortic wall of mice exposed to hypobaric hypoxia may stabilize the vascular wall by inhibition of microvascular sprouting. The surprising finding of increased endostatin in the lung presumably contributes to the development of pulmonary hypertension by reduction of angiogenesis.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Relationship between protozoan and metazoan communities and operation and performance parameters in a textile sewage activated sludge system

The present study aims at investigating the possibility of assessing performance and depuration conditions of an activated sludge wastewater treatment plant through an exploration of the microfauna. The plant, receiving textile industrial (70%) and domestic (30%) sewage, consists of a two-step biological depurating plant, with activated sludge followed by a percolating system. A total of 35 samples were analyzed during five months, and 30 taxa of protozoa and small metazoa were found. Epistylis rotans, Vorticella microstoma, Aspidisca cicada and Arcella sp. were the most frequent protozoa identified. Several significant correlations between biological, physical–chemical and operational parameters were determined, but no significant correlations could be established between biological parameters and removal efficiencies. The Sludge Biotic Index (SBI) reflected the overall state of the community but only presented statistically significant correlations with the influent total suspended solids (TSS), total suspended solids in mixed-liquor (MLTSS) and dissolved oxygen (DO). The determination of key groups and taxa along with general community parameters showed to have potential value as indicators of the depuration conditions. Despite the impossibility of correlating biological parameters and the removal efficiencies, the present study attests the value of the microfauna to assess the operation of the activated sludge systems even in the case of non-conventional plants and/or plants receiving industrial sewage.     

Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats

Hyperglycemia induced oxidative stress has been proposed as a cause of many complications of diabetes including cardiac dysfunction. The present study depicts the therapeutic effect of green tea extract on oxidative stress in aorta as well as heart of streptozotocin diabetic rats. Six weeks after diabetes induction, green tea was administered orally for 4 weeks [300 mg (kg body weight)(-1) day (-1)]. In aorta and heart of diabetic rats there was a significant increase in the activity of superoxide dismutase, catalase and glutathione peroxidase with an increase in lipid peroxides. Diabetic rats showed a significant decrease in the levels of serum and cardiac glutathione. Green tea administration to diabetic rats reduced lipid peroxides and activity of antioxidant enzymes whereas increased glutathione content. The results demonstrate that the induction of antioxidant enzymes in diabetic rats is not efficient and sufficient to reduce the oxidative stress. But green tea by providing a competent antioxidative mechanism ameliorates the oxidative stress in the aorta and heart of diabetic rats. The study suggests that green tea may provide a useful therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes mellitus.     

The ultrastructure of basal cells of rat and dog prostate

Summary The ultrastructure of the basal cells of rat lateral and ventral prostate and of dog prostate has been studied. Basal cells from both species appear as undifferentiated cells, characterised by a lack of cytoplasmic organelles and a poorly developed Golgi complex and endoplasmic reticulum. The presence of cytoplasmic filaments and micropinocytosis is not considered to be sufficient evidence to assume any similarity to myoepithelium, as has been previously suggested. Basal cells are instead considered to be precursors of secretory epithelial cells.The authors wish to express their gratitude to the Tenovus Organisation for their generous financial support. This work was also supported by a grant from the Medical Research Council No. G974/304B. One of them (FS) was supported by the British Council