The antiatherogenic effect of new biocompatible cationically modified polysaccharides: chitosan and pullulan - the comparison study.

Pullulan and chitosan are biocompatible polysaccharides obtained from natural sources with many biomedical applications. Cationically modified polymers, such as chitosan and pullulan after covalent attachment of glycidyltrimethylammonium chloride (GTMAC), showed beneficial biological properties. In the present study, it was clearly demonstrated and confirmed that both cationically modified polysaccharides (chitosan-GTMAC and pullulan-GTMAC) have the antiatherosclerotic potential by inhibition of atherosclerotic plaque development and controlling the expression of genes involved in lipid metabolism. It has also been shown that the cationically modified chitosan (HTCC) at a dose of 200 mg/kg b.w./day in male apoE-knockout mice acted as hypolipidaemic agent. It was observed that a statistically significant decrease in low-density lipoprotein (LDL) cholesterol level by 32% occurred under the influence of HTCC at a dose of 200 mg/kg b.w./day after 16 weeks of the experiment compared to the control group of apoE(-/-) mice. Moreover, under the influence of cationically modified chitosan administered orally to female apoE-knockout mice at a dose of 300 mg/kg b.w./day for 18 weeks a statistically significant reduction by 33% in the area of atherosclerotic plaque was observed compared to the control group, i.e., apoE-knockout mice whose diet was not supplemented with the cationically modified polysaccharide. Current in vivo studies connected with cationically modified pullulan showed a statistically significant 22% reduction of the area of atherosclerotic plaque in the apoE(-/-) mice fed with a feed containing Pull-GTMAC at a dose of 500 mg/kg b.w./day for 18 weeks in comparison to the control group of apoE-knockout mice. In the in vitro studies it was also shown that cationically modified chitosan acted therapeutically by reduction of the level of the expression of human 3-hydroxy-3-methylglutaryl-CoA reductase (human HMG-CoAR) after 24 hours of incubation with HepG2 cells. However, cationically modified pullulan did not show this effect in the experiment on HepG2 cell line. On the other hand, Pull-GTMAC caused a statistically significant increase in insulin induced gene 1 (INSIG1) expression and increase in mRNA level of LDL receptor in brown fat tissue of female apoE-knockout mice after oral administration with feed at a dose of 300 mg/kg b.w./day for 18 weeks in comparison to the control group of apoE(-/-) mice, that was crearly demonstrated the effect of cationically modified pullulan on the expression of lipid metabolism genes in in vivo conditions. In the present article we have shown for first time that cationically modified pullulan and chitosan have some similarities in their antiatherogenic action but there are also some minor differences in mechanism of their effect on lipid metabolism.

Anti-atherosclerotic effect of the angiotensin 1-7 mimetic AVE0991 is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis.

BACKGROUND AND PURPOSE: Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1-7 (Ang-(1-7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1-7) mimetics, such as AVE0991. The mechanisms underlying the vaso-protective effects of AVE0991, a Mas receptor agonist, remain to be explored. EXPERIMENTAL APPROACH: We investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)-/- mice, in the context of vascular inflammation and plaque stability. KEY RESULTS: AVE0991 has significant anti-atherosclerotic properties in ApoE-/- mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoE-/- mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1ß, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. CONCLUSIONS AND IMPLICATIONS: The selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoE-/- mice. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

New cationically modified pullulan attenuates atherogenesis and influences lipid metabolism in apoE-knockout mice.

Pullulan is a biocompatible polysaccharide obtained from black, yeast-like fungus Aureobasidium pullulans. This polymer is used to deliver various substances to the liver because of its specificity for this organ. Pullulan is internalized into hepatocytes in the process of asialoglycoprotein receptor mediated endocytosis. Recently, by reaction with glycidyltrimethylammonium chloride (GTMAC) we have successfully synthesized a cationically-modified pullulan (Pull-GTMAC). Pull-GTMAC exhibits some unique beneficial effects not found for its native counterpart. In this article we have reported for the first time that Pull-GTMAC administered orally to apoE-knockout mice (murine model of atherosclerosis) at a dose of 300 mg/kg b.w./day for 18 weeks showed anti-atherosclerotic activity reducing the area of atherosclerotic plaque. We have also found that Pull-GTMAC at a dose of 300 mg/kg b.w./day increases both the average daily mass of feces and the average number of droppings excreted by apoE(-/-) mouse in relation to the control sample derived from the mice fed with feed without the tested compound. However, the raw fat content in the feces of apoE-knockout mice was decreased in the group fed with the diet containing Pull-GTMAC towards control group of animals. Pull-GTMAC caused also statistically significant increase of mRNA level for LDL receptor in the apoE(-/-) mice liver after administration at a dose of 300 mg/kg/b.w./day for 18 weeks. However, the compound had no impact on lipid profile in serum of the tested mice. What is more, the studies on HepG2 cell line indicated an antiproliferative potential of cationically modified pullulan after 24 hour and 48 hour of incubation with the polysaccharide. In this paper we have shown for first time that cationically modified pullulan has antiatherogenic potential and influences on lipid metabolism.

Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 µM), thiorphan (3 µM), or vehicle and incubated for 15 minutes with ANG I (1 µM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1-9), ANG (1-7), and ANG (1-5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1-9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1-7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1-9), and ANG (1-7)) ANG I metabolites.

Polymorphism in the chemokine receptor 7 gene (CCR7) is associated with previous myocardial infarction in patients undergoing elective coronary angiography.

Coronary artery disease (CAD) remains a major cause of death in developed countries. Both environmental and, less known, genetic factors contribute to progression of CAD to myocardial infarction (MI). Immune system is activated in patients with CAD through dendritic cells (DCs), which present plaque antigens to T lymphocytes. Production of proinflammatory cytokines by activated T cells contributes to plaque rupture in MI. Chemokine receptor 7 (CCR7) on DCs is required for their chemotaxis from plaque to lymph nodes. This makes possible an interaction of DCs with T lymphocytes and initiation of specific immune response. We hypothesized that single nucleotide polymorphisms (SNPs) in CCR7 gene locus are associated with previous MI in patients with CAD. To test this hypothesis, we genotyped six SNPs from the CCR7 gene locus in 300 consecutive patients, admitted for elective coronary angiography. We performed univariate-, multivariate- (including potential confounders) and haplotype-based tests of association of SNPs with previous MI and results of angiography. Allele A of rs17708087 SNP was associated with previous MI. This association remained significant after adjustment for age, sex, smoking, hypercholesterolaemia and drugs used by patients (odds ratio 2.13, 95% confidence interval: 1.13-3.86). Therefore, we conclude that CCR7 gene locus harbours a polymorphism that modifies risk of MI in patients with CAD. Replication of this association could be sought in a prospective cohort of initially healthy individuals.

The renin-angiotensin system and its vasoactive metabolite angiotensin-(1-7) in the mechanism of the healing of preexisting gastric ulcers. The involvement of Mas receptors, nitric oxide, prostaglandins and proinflammatory cytokines.

The inhibition of angiotensin-converting enzyme (ACE) or the blockade of angiotensin (Ang) AT-1 receptors affords protection against acute gastric mucosal injury, but whether the major metabolite of renin-angiotensin system (RAS), Ang-(1-7), accelerates the healing process of preexisting gastric ulcers remains unknown. Previous studies documented that Ang-(1-7) acting via its own Mas receptor exerts vascular responses opposing those of Ang II. We studied the effects of the Ang-(1-7)/Mas receptor axis on the healing rate of acetic-acid-induced gastric ulcers with or without the blockade of Mas receptors by A 779 and compared it with the effects of activation and blockade of the AT-1 receptor by the treatment with Ang II and losartan, respectively, the inhibition of ACE by lisinopril, the NO/cNOS inhibition by L-NAME and inhibition of prostaglandin/COX system by indomethacin in the presence of Ang-(1-7). Additionally, ex vivo metabolism of Ang I in gastric tissue was assessed by LC/MS method. At day 9 after ulcer induction, the area of these ulcers and the accompanying changes in total gastric blood flow (GBF) were determined as were gastric mucosal blood flow (GMBF) at ulcer margin and gastric oxygen uptake (GVO2). The gastric mucosal expression of mRNAs for constitutive nitric oxide synthase (cNOS), superoxide dismutase (SOD), and pro-inflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) and plasma level of both cytokines were determined by RT-PCR and ELISA. The 9 days treatment with Ang II dose-dependently increased the area of gastric ulcers and this effect was accompanied by a significant fall in the GBF, GVO2 and GMBF at ulcer margin. In contrast, treatment with Ang-(1-7) which produced a significant rise in the luminal content of NO significantly reduced the area of gastric ulcer and significantly increased the GBF, GVO2 and the GMBF at ulcer margin. Similar GMBF changes and significant reduction the area of gastric ulcer was observed in rats with gastric ulcers treated with the agonist of Mas receptor, AVE 0991. These effects of Ang-(1-7) and AVE 0991 were eliminated by blockade of the Mas receptor with A779. Similarly to Ang-(1-7), treatment with losartan or lisinopril significantly reduced the area of gastric ulcers and the accompanying increase in the GMBF at ulcer margin and these effects were significantly attenuated by a concomitant administration of L-NAME and indomethacin. The rate of healing of ulcers was associated with a decrease in ex vivo Ang-(1-7) formation and this effect was attenuated by lisinopril. The treatment with Ang-(1- 7) or AVE 0991 increased the expression of mRNA for cNOS and SOD and downregulated that of IL-1ß and TNF-α followed by the decrease in the plasma IL-1ß and TNF-α levels. We conclude that the Ang-(1-7)/Mas receptor system accelerates the healing of preexisting gastric ulcers via an increase in the gastric macro- and microcirculations, and an increase in gastric tissue oxygenation. These effects are mediated by PG and NO derived from overexpression of cNOS, an increase in the expression of antioxidizing enzyme SOD 2 and an anti-inflammatory action involving the inhibition of expression and release of pro-inflammatory cytokines IL-1ß and TNF-α. Our results seem to underlie the importance of the Ang-(1-7), AT-1 and Mas receptors in the regulation of local vascular and metabolic effects associated with mechanism of gastric ulcer healing.

Perindoprilat changes ANG (1-9) production in renal arteries isolated from young spontaneously hypertensive rats after ANG I incubation.

We used mass spectrometry to quantitate production of angiotensinogen metabolites in renal artery of 3- and 7-month-old Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Tissue fragments were incubated for 15 min in oxygenated buffer, with added angiotensin I. Concentrations of angiotensins I (ANG I), II (ANG II), III (ANG III), IV (ANG IV), angiotensin (1-9) [ANG (1-9)], angiotensin (1-7) [ANG (1-7)], and angiotensin (1-5) [ANG (1-5)], excreted into the buffer during experiment, were measured using liquid chromatography-mass spectrometry (LC/MS) and expressed per mg of dry tissue. Effects of pretreatment with 10 microM perindoprilat on the production of ANG I metabolites were quantitated. Background production of any of ANG I metabolites differed neither between WKY and SHR rats nor between 3- and 7-month-old rats. Perindoprilat pretreatment of renal arteries resulted, as expected, in decrease of ANG II production. However, renal arteries of 7-month-old SHR rats were resistant to ACE inhibitor and did not change ANG II production in response to perindoprilat. In renal arteries, taken from 3-month-old rats, pretreated with perindoprilat, incubation with ANG I, resulted in the level of ANG (1-9) significantly higher in SHR than WKY rats. Our conclusion is that in SHR rats, sensitivity of renal artery ACE to perindoprilat inhibition changes with age.

Third-stage nematode larvae of Contracaecum osculatum from Baltic cod (Gadus morhua) elicit eosinophilic granulomatous reactions when penetrating the stomach mucosa of pigs.

Third-stage larvae of the anisakid nematode Contracaecum osculatum were recovered from livers of Atlantic cod (Gadus morhua) caught in the Baltic Sea (June 2014) and used for experimental infection of two pigs (one male and one female). Each pig received 215 larvae by oral infection (feeding with minced cod liver containing live nematode larvae). Pigs were euthanized after 5 days, necropsied, and subjected to parasitological investigation. A total of 12 larvae were found penetrating the mucosa of the ventricle (7 in the female pig and 5 in the male pig) eliciting a granulomatous reaction at the penetration site. Four non-attached larvae were found in the female pig stomach and one in the male pig. Petechial bleeding was observed at several locations in the ventricular mucosa where larvae were located. Histological examination of the stomach mucosa revealed a massive cellular infiltration (giant cells, lymphocytes, macrophages, granulocytes, and fibroblast like cells) around the penetrating larva. Mononuclear and polymorphonuclear cells containing eosinophilic granulae were particularly prominent in the granulomas. Reactions correspond to reactions in pigs following experimental infection with the human pathogenic anisakid larvae Anisakis sp. and Pseudoterranova sp. which suggests that C. osculatum might have a zoonotic potential as well.

The current view on biological potency of cationically modified chitosan.

Chitosan is biocompatible polymer obtained from chitin, the building component of the crustacean shells. In this paper we make an attempt to review the current state of knowledge on some biological effects of chitosan in comparison with those of cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) that was recently synthetized by us by covalent attachment of glycidyltrimethylammonium chloride (GTMAC). Biological effects of HTCC and non-modified polymer are very similar. However, HTCC shows some unique beneficial properties which have not been found in its non-modified counterpart. One such example is that HTCC has the ability to bind heparin at physiological pH. HTCC having the degree of substitution almost 63.6% is easily absorbed within 1 hour after oral administration as found in C57BL/6j mice using FITC-labeled polymer. HTCC is distributed to lung, heart, and kidneys. HTCC stimulates and enhances blood platelet aggregation and decreases erythrocyte deformability (RBC). Moreover, HTCC seems to decrease both plasma total cholesterol level and LDL-cholesterol level in apoE-knockout mice fed with a diet containing HTCC. HTCC possibly down-regulates the HMG-CoAR mRNA level after 24 hour incubation with HepG2 cells in vitro.

Significant deterioration of anti-atherogenic efficacy of nebivolol in a double (apolipoprotein E and endothelial nitric oxide synthase) knockout mouse model of atherosclerosis in comparison to single (apolipoprotein E) knockout model.

Anti-atherogenic action of nebivolol in apolipoprotein E (apoE)-single knockout mouse model can be explained by its beneficial effect on endothelium, especially on endothelial nitric oxide synthase (eNOS). We, therefore, decided to use apoE and eNOS-double knockout mouse model to confirm that mechanism of nebivolol beneficial action. In apoE-single knockout mice, lesion area measured by "cross-section" of aortic roots was 79,244 ± 6,143 µm(2) in the control group versus 65,347 ± 6,152 µm(2) in nebivolol-treated group (P<0.05). However, in apoE and eNOS-double knockout mice, lesion area measured by "cross-section" of aortic roots was 92,319 ± 8,876 µm(2) in the control group versus 98,609 ± 9,164 µm(2) in nebivolol-treated group (P>0.05). The comparison between apoE-single knockout mice and apoE & eNOS-double knockout mice without treatment also showed statistically significant difference: 81,232 ± 8,264 µm(2) versus 92,319 ± 8,876 µm(2) (P<0.05). This is the first report that describes the effect of nebivolol on atherogenesis in apoE and eNOS-double knockout mice, proving directly the necessity of the presence of eNOS in endothelium for nebivolol to show its an anti-atherogenic potency.

The influence of angiotensin-(1-7) peptidomimetic (AVE 0991) and nebivolol on angiotensin I metabolism in aorta of apoE-knockout mice.

The detrimental role of over activation of renin-angiotensin system (RAS) in atherogenesis is widely recognized. Recently, we have demonstrated that Ang-(1-7) peptidomimetic - AVE0991, as well as known beta-adrenolytic agent nebivolol, exert anti-atherogenic actions in mouse model of atherosclerosis - apoE-knockout mice. Here, using LC-ESI-MS ex vivo system, we tested whether prolonged treatment of apoE-knockout mice by these drugs can influence RAS in aorta of apoE-knockout mice in regard to generation of most active metabolites of Ang I-Ang II and Ang-(1-7). As compared to wild type animals there was increased generation of Ang II in aorta of apoE-knockout mice, while the formation of Ang-(1-7) did not differ between both groups. Either treatment with AVE0991 or nebivolol resulted in significant attenuation of Ang II production in aorta of apoE-knockout mice. In conclusion, for the first time we directly demonstrated that there is increase in ability of aortic tissue to generate Ang II in mouse model of atherosclerosis of apoE knockout mice, and that such effect could be efficiently attenuated either by treatment of nebivolol or Ang-(1-7) peptidomimetic - AVE0991. The exact mechanism(s) responsible for interference of both drugs with RAS require further investigation.

Vasodilatory effect and endothelial integrity in papaverine- and milrinone-treated human radial arteries.

Prevention of the vasospasm is an important aspect of coronary artery bypass grafting (CABG) with the use of radial artery (RA) as the conduit. We compared the effect of two phosphodiesterase inhibitors papaverine and milrinone on vasodilation and endothelial integrity of human RA segments harvested from 20 CABG patients. Vasodilatory effect of the drugs were assessed by organ bath technique in RA rings precontracted with KCl and phenylephrine. Endothelial integrity was evaluated by CD34 immunofluorescence in frozen sections. Vasorelaxation induced by papaverine was significantly greater as compared to that induced by milrinone (90.47% ± 10.16% vs. 78.98% ± 19.56%, p<0.05). Similarly, pretreament with papaverine more strongly inhibited the contractile response of RA rings to KCl (6.0 ± 8.0 mN vs. 26.7 ± 21.5 mN, p<0.001). Papaverine was also superior to milrinone in the preservation of endothelial integrity (75.3% ± 12.9% vs. 51.8% ± 18.0%, p<0.02). In conclusion, papaverine seems to be more suitable than milrinone for prevention of vasospasm in radial artery conduits used for CABG.

Prolactin--not only lactotrophin. A "new" view of the "old" hormone.

Prolactin (PRL) is a hormone mainly secreted by the anterior pituitary. Recent studies have shown that it may also be produced by many extrapituitary cells. The PRL gene expression is controlled by two independent promoter regions, which may be differentially regulated in the pituitary and extrapituitary organs. Proteolytic modifications of PRL generate variants of the hormone. A16 kDa PRL fragment, acting through a specific receptor, has both an antiangiogenic activity as well as an inhibitory effect on tumor growth. Stimulation of the PRL receptor involves many signal transduction pathways, for example JAK2/STAT, MAPK, c-src and Fyn kinase cascade, and these pathways may vary in different tissues. PRL synthesis and secretion is mainly regulated by the inhibitory influence of dopamine but other hormones are also involved in these mechanisms. The essential biological action of PRL is the stimulation of lactogenesis and galactopoesis. Apart from its classical functions, PRL affects other aspects of human body function including osmoregulation, metabolism and regulation of the immune and the central nervous system. Hyperprolactinemia is a common syndrome affecting both men and women. It is manifested by the presence of galactorrhoea and through the symptoms of hypogonadotrophic hypogonadism. Following on from the fact that PRL has so many pleiotropic tissue specific effects it is not surprising to learn that hyperprolactinaemia is a systemic condition which may predispose to numerous cardiovascular and immune-mediated reactions. The exact effects of PRL on both immune and cardiovascular systems are being currently unraveled and may lead to the introduction of novel therapeutic approaches in the future.

Angiotensin-(1-7) receptor Mas agonist ameliorates progress of atherosclerosis in apoE-knockout mice.

Our interest focused on an open question whether AT-(1-7), nonpeptide receptor agonist: AVE 0991, is able to ameliorate atherosclerosis. We used an apolipoprotein E (apoE) - knockout mice model of atherosclerosis. Experimental groups received the same diet as control, mixed with: AVE 0991 at a dose of 0.58 µmol/kg b.w./day, perindopril at a dose of 0.4 mg/kg b.w./day or with tiorphan at a dose of 2.5 mg/kg b.w./day. A-779 [(D-alanine)-angiotensin (1-7)] was given at a dose of 3.3 mg/kg b.w., 3 times a week i.p. Measured by "en face" method, the percentage of occupied by Sudan IV-stained surfaces were as follows: 14.2±1.9 % in control group, whereas in AVE 0991-treated as well as in perindopril-treated groups percentages were statistically significantly lower. In tiorphan group there was no change comparing to control group, whereas in A-779 group percentage was statistically significantly higher. "Cross-section" of aortic roots revealed also the difference in atherosclerotic lesions. The mean surfaces, occupied by oil red O-stained changes were: 91.213±8.123 µm(2) in control group, while in AVE 0991-treated as well as in perindopril-treated groups lesions were statistically significantly lower. In tiorphan group there was no change; however, in A-779 group lesions were statistically significantly higher. Measured by real time RT-PCR relative p22phox (submit of NADPH oxidase) expression was significantly decreased in AVE 0991-treated mice. As revealed by flow cytometry, the expression of co-stimulatory molecules: CD86, CD80 and CD40 on both dendritic cells (CD11c+) and macrophages (F4/80+) was reduced in AVE 0991-treated group, which correlated with decreased expression of CD69 activation marker on CD4+T cells. In our report we showed the beneficial effect of AVE 0991 on atherogenesis in gene-targeted mice.

Novel concept in the mechanism of injury and protection of gastric mucosa: role of renin-angiotensin system and active metabolites of angiotensin.

The term cytoprotection pioneered by Robert and colleagues has been introduced to describe the remarkable ability of endogenous and exogenous prostaglandins (PGs) to prevent acute gastric hemorrhagic lesions induced by noxious stimuli such as ethanol, bile acids, hiperosmolar solutions and nonsteroidal anti-inflammatory agents such as aspirin. Since that time many factors were implicated to possess gastroprotective properties such as growth factors including epidermal growth factor (EGF) and transforming factor alpha (TGFα), vasodilatory mediators such as nitric oxide (NO) and calcitonin gene related peptide (CGRP) as well as appetite gut hormones including gastrin and cholecystokinin (CCK), leptin and recently ghrelin. This protective action of gut peptides has been attributed to the release of PG but question remains whether another peptide angiotensin, the classic component of the systemic and local renin-angiotensin system (RAS) could be involved in the mechanism of gastric integrity and gastroprotection. After renin stimulation, the circulating angiotensin I is converted to angiotensin II (ANG II) by the activity of the Angiotensin Converting Enzyme (ACE). The ANG II acting via its binding to two major receptor subtypes the ANG type 1 (AT1) and type 2 (AT2) has been shown be activated during stress and to contribute to the pathogenesis of cold stress- and ischemia-reperfusion-induced gastric lesions. All bioactive angiotensin peptides can be generated not only in systemic circulation, but also locally in several tissues and organs. Recently the new functional components of RAS, such as Ang-(1-7), Ang IV, Ang-(1-12) and novel pathways ACE2 have been described suggesting the gastroprotective role for the novel ANG II metabolite, Ang-(1-7). The fact that Ang-(1-7) is produced in excessive amounts in the gastric mucosa of rodents and that pretreatment by Ang-(1-7) exhibits a potent gastroprotective activity against the gastric lesions induced by cold-restraint stress suggests that this and possibly other vasoactive metabolites of ANG II pathway could be involved in the mechanism of gastric integrity and gastroprotection. This review summarizes the novel gastroprotective factors and mechanisms associated with metabolic fate of systemic and local RAS activation with major focus to recent advancement in the angiotensin pathways in the gut integrity.

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ).

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.

The effect of doxycycline on atherogenesis in apoE-knockout mice.

Doxycycline at subantimicrobial doses inhibits matrix metalloproteinases (MMPs) activity, and is the only MMP inhibitor which is widely available in clinical practice. The aim of the study was to reveal whether non-specific MMPs inhibition by tetracycline could ameliorate development of atherosclerosis in apolipoprotein E (apoE)-knockout mice. Doxycycline (1.5 mg/ kg b.w./day) administered orally attenuated atherogenesis, measured both by "en face" method (10.25±1.7% vs. 15.7±2.0%, p<0.05) and "cross-section" method (66,254±7,468 µm(2) vs. 90,687±8,521 µm(2), p<0.05). In-situ zymography showed decrease of the extent of non-specific gelatinase activity in doxycycline-treated mice This is the first report to date describing the effect of doxycycline on atherogenesis in apoE-targeted mice.

Evolution of rheological properties of erythrocytes and left ventricular geometry in cardiovascular disease risk patients.

The evolution of rheological properties of erythrocytes and geometrical parameters of left ventricle during therapies aimed at reducing cardiovascular disease (CVD) risk has been investigated. The study group consisted of 29 individuals who were diagnosed with the presence of at least one CVD risk factor at the time of entry to the study. Appropriate therapies were applied and the patients were followed for two years. Two groups of patients could be distinguished. The first group consisted of 12 individuals who were rigorously applying the therapy and for whom blood pressure, total cholesterol, LDL and glucose returned to normal levels. The second group included 17 patients for whom the above mentioned parameters remained pathological in spite of the applied therapy. In the first group, erythrocyte deformability as well as LVMI improved: deformability increased on average by 17% (p < 0.025), whereas LVMI decreased by 8% but not in a statistically significant manner (p < 0.27). In the second group, the results indicate worsening of both hemorheological properties and left ventricular geometry: RBC deformability became lower by 15% (p < 0.00001) and LVMI increased by 18% although this change was not statistically significant (p < 0.19). The results indicate that blood rheology improves when the CVD risk is reduced by administered therapy and worsens when the risk increases. Similar behavior shows LVMI. It is very likely that left ventricular geometry is influenced by blood rheology.

AVE 0991-angiotensin-(1-7) receptor agonist, inhibits atherogenesis in apoE-knockout mice.

Recent evidence shows that the renin-angiotensin system is a crucial player in atherosclerotic processes. It was also proved that Ang II promotes atherogenesis. Angiotensin-(1-7) [Ang-(1-7)] opposites Ang II action. Therefore, we would like to find out whether Ang-(1-7) receptor agonist: AVE 0991, could ameliorate atherosclerosis progression in an experimental model of atherosclerosis: apolipoprotein E (apoE) - knockout mice. AVE 0991 inhibited atherogenesis, measured both by "en face" method (7.63+/-1.6% vs. 14.6+/-2.1%) and "cross-section" method (47 235+/-7 546 microm(2) vs. 91 416+/-8 357 microm(2)). This is the first report showing the effect of AVE 0991 on atherogenesis in gene-targeted mice.

The current view on the role of leukotrienes in atherogenesis.

Since inflammation plays an important role in atherogenesis, during recent years it has become apparent that the 5-lipoxygenase (5-LO) pathway may take significant part in modifying the pathogenesis of atherosclerosis. Indeed, it has been recently demonstrated that the 5-LO substantially contribute to atherosclerosis in both mouse models and humans. However, animal models potentially bear the risk of compensatory mechanisms, due to genetic modification of the target gene that render the results difficult to interpret. Another caveat is species differences between mice and humans. 5-LO expression in intimal atherosclerotic lesions varies between mice and humans; also, 5-LO and 12/15-LO appear to be differentially regulated in inflammatory cells of mice. Moreover, atherogenesis in mice differs in several facets from the human pathology. Thus, T cells, whose presence in all stages of atherosclerotic lesions is acknowledged, are underrepresented in murine models of atherosclerosis. 5-LO/LT pathway shows important disparities between murine and human atherosclerosis. Advanced human plaques show differences in 5-LO expression compared with mouse lesions. Taken together, in advanced human atherosclerosis, a role for 5-LO is likely, which is distinct from its role in early atherogenesis. This presence of the 5-LO/LT pathway in advanced lesions is not found in mouse models, which might be due to: (i) rapid progression of atheroma growth in mice vs. slower, often interrupted progression in humans; (ii) advanced human plaques display a higher degree of instability and risk to rupture than murine plaques; (iii) temporal dissociation in the Th1/Th2 'balance' at distinct lesion stages between mice and humans.