Endothelin and diabetic complications: a brain-centric view.

The global epidemic of diabetes is of significant concern. Diabetes associated vascular disease signifies the principal cause of morbidity and mortality in diabetic patients. It is also the most rapidly increasing risk factor for cognitive impairment, a silent disease that causes loss of creativity, productivity, and quality of life. Small vessel disease in the cerebral vasculature plays a major role in the pathogenesis of cognitive impairment in diabetes. Endothelin system, including endothelin-1 (ET-1) and the receptors (ET(A) and ET(B)), is a likely candidate that may be involved in many aspects of the diabetes cerebrovascular disease. In this review, we took a brain-centric approach and discussed the role of the ET system in cerebrovascular and cognitive dysfunction in diabetes.

Endothelin-1 inhibition improves the mBDNF/proBDNF ratio in endothelial cells and HT22 neurons under high glucose/palmitate growth conditions.

Diabetes increases the risk and worsens the progression of cognitive impairment. The hippocampus is an important domain for learning and memory. We previously showed that endothelin-1 (ET-1) reduced diabetes-induced inflammation in hippocampal neurons, suggesting a neuroprotective effect. Given that neurons and endothelial cells within the neurovascular unit depend on each other for proper function, we investigated the effect of ET-1 on brain-derived neurotrophic factor (BDNF) synthesis, a key neurotrophin and prosurvival factor, in neuronal (HT22 hippocampal neurons) and brain microvascular endothelial (BMEC-5i) cells under normal and diabetes-mimicking (high glucose plus palmitate) conditions. Cells were treated with exogenous ET-1 or ET receptor antagonists including ET(B) receptor selective antagonist BQ788 (1 microM) or dual-receptor antagonist bosentan (10 microM). Mature (m)BDNF, proBDNF and caspase-3 levels were measured by Western blotting. Diabetic conditions reduced the prosurvival mBDNF/proBDNF ratio in both HT22 and BMEC-5i cells. Addition of exogenous ET-1 had no effect on the BDNF system in HT22 cells in diabetic conditions. Both HT22 and BMEC-5i cells had an increase in the mBDNF/proBDNF ratio when grown in diabetes-simulating conditions in the presence of endothelin receptor inhibition. These data suggest that blockade of ET-1 may provide neuroprotection to hippocampal cells through the modulation of the BDNF system.

Lipopolysaccharide-induced necroptosis of brain microvascular endothelial cells can be prevented by inhibition of endothelin receptors.

Over activation of the endothelin-1 (ET-1) system in disease states contributes to endothelial dysfunction. On the other hand, ET-1 promotes proliferation and survival of endothelial cells. Regulation of programmed cell death (PCD) pathways is critical for cell survival. Recently discovered necroptosis (regulated necrosis) is a pathological PCD mechanism mediated by the activation of toll like receptor 4 (TLR4), which also happens to stimulate ET-1 production in dendritic cells. To establish the effect of ET-1 on PCD and survival of human brain microvascular endothelial cells (BMVECs) under control and inflammatory conditions, BMVECs were treated with ET-1 (10 nM, 100 nM and 1 microM) or lipopolysaccharide (LPS, 100 ng/ml). ET receptors were blocked with bosentan (10 microM). Under normal growth conditions, exogenous ET-1 reduced BMVEC viability and migration at a relatively high concentration (1 microM). This was accompanied with activation of necroptosis and apoptosis marker genes. LPS decreased endogenous ET-1 secretion, increased ET(B) receptor expression and activated necroptosis. Even though ET-1 levels were low (less than 10 nM levels used under normal growth conditions), blocking of ET receptors with bosentan inhibited the necroptosis pathway and improved the cell migration ability of BMVECs, suggesting that under inflammatory conditions, ET-1 activates PCD pathways in BMVECs even at physiological levels.

Reduction in serum paraoxonase level in newborns with hyperbilirubinemia as a marker of oxidative stress.

OBJECTIVE: Indirect bilirubin exerts an antioxidant effect when increased mildly. This study aimed to investigate whether increased bilirubin levels lead to an oxidant effect in newborns with hyperbilirubinemia requiring phototherapy. PATIENTS AND METHODS: The study included 30 term newborn infants aged 0-7 days with indirect hyperbilirubinemia requiring phototherapy and no comorbid disease as the study group. In addition, 30 term healthy newborn infants aged 0-7 days without indirect hyperbilirubinemia were employed as a control group. Serum triglyceride, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), serum paraoxonase (PON) levels and malondialdehyde (MDA) levels were compared between the groups. RESULTS: Serum MDA, total bilirubin, and LDL and HDL levels were significantly higher and the serum PON level was significantly lower, in the study group compared with the controls (p < 0.05). CONCLUSION: In newborns with hyperbilirubinemia requiring phototherapy, an increased bilirubin level causes oxidative stress by decreasing the level of serum PON and increasing the level of MDA.

Tumour necrosis factor-alpha, interleukin-10, interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta.

No data exist to assess certain polymorphisms that have a potential effect on the immune response in patients with chronic hepatitis delta (CHD). The aim of this study was to investigate polymorphisms in 6 polymorphic sites: IL-10 -1082 (rs1800896), IL-10 -627 (rs1800872), IFN-γ +874 (rs62559044), TNF-α -308 (rs1800629), vitamin D receptor (VDR) FokI (rs2228570) and VDR TaqI (rs731236). The genotypes of 67 patients with CHD and 119 patients with chronic hepatitis B (CHB) were compared. In addition, 56 individuals with resolved hepatitis B virus (HBV) infection were used as a control group for patients with CHB. Polymorphisms in TNF-α, IL-10, and VDR genes were analysed using polymerase chain reaction/restriction fragment length polymorphism methods. The IFN-γ gene polymorphism was detected by allele-specific polymerase chain reaction (PCR). Patients with CDH were more likely to have advanced liver disease compared with patients with CHB (P < 0.0001). IL-10 -1082 and VDR TaqI polymorphisms showed significant differences between patients with CHD and CHB. The high secretory IL-10 -1082 genotype GG was less frequent in CHD compared with patients with CHB and resolved HBV (17.7%, 37.4% and 47.1%, respectively (P < 0.05 for CHD vs CHB and resolved HBV). The frequency of the high secretory VDR TaqI TT genotype was 86.6% in patients with CHD, 62.7% in patients with CHB and 62.5% in resolved HBV individuals (CHD vs CHB: P < 0.05). None of the polymorphisms analysed had an effect on HBV persistence. IL-10 -1082 and VDR TaqI polymorphisms may contribute to the more severe liver disease associated with CHD compared with CHB.

Molecular analysis of East Anatolian traditional plum and cherry accessions using SSR markers.

We conducted SSR analyses of 59 accessions, including 29 traditional plum (Prunus domestica), 24 sweet cherry (Prunus avium), and 1 sour cherry (Prunus cerasus) selected from East Anatolian gene sources and 3 plum and 2 cherry reference accessions for molecular characterization and investigation of genetic relationships. Eight SSR loci [1 developed from the apricot (UDAp-404), 4 from the peach (UDP96-010, UDP96-001, UDP96-019, Pchgms1) and 3 from the cherry (UCD-CH13, UCD-CH17, UCD-CH31) genome] for plum accessions and 9 SSR loci [5 developed from the cherry (PS12A02, UCD-CH13, UCD-CH17, UCD-CH31, UCD-CH21), 3 from the peach (Pchgms1, UDP96-001, UDP96-005) and 1 from the plum (CPSCT010) genome] for cherry accessions were used for genetic identification. A total of 66 and 65 alleles were obtained in the genetic analyses of 31 plum and 28 cherry accessions, respectively. The number of alleles revealed by SSR analysis ranged from 4 to 14 alleles per locus, with a mean value of 8.25 in plum accessions, and from 5 to 10 alleles per locus with a mean value of 7.2 in cherry accessions. Only one case of synonym was identified among the cherry accessions, while no case of synonym was observed among the plum accessions. Genomic SSR markers used in discrimination of plum and cherry accessions showed high cross-species transferability in the Prunus genus. Because of their appreciable polymorphism and cross species transferability, the SSR markers that we evaluated in this study will be useful for studies involving fingerprinting of cherry and plum cultivars.

Characterization of Anatolian traditional quince cultivars, based on microsatellite markers.

We conducted simple sequence repeat (SSR) analyses of 15 traditional quince (Cydonia oblonga) cultivars from Anatolian gene sources for molecular characterization and investigation of genetic relationships. Three pear and two apple cultivars were used as references for SSR locus data analysis and to determine allele profiles between species. Eight SSR loci that were developed from apple and pear were used, and a total of 44 alleles were found among quince cultivars. The CH01F02 locus was found to have the highest identification probability, while the CH04E03 locus had the lowest identification probability. Analysis of similarity ratios between quince cultivars showed that the lowest similarity ratio was 18% (Esme-Bardacik ± k), while the highest similarity ratio was 87% (Bursa-Osmancik ± k and Osmancik ± k-Viranyadevi). In the phylogenetic dendrogram, Esme quince showed separate branching from other quince cultivars, and no synonymous accessions were found. These results suggest that SSR markers from pear and apple could be used to determine genetic variation among quince cultivars. These findings can be used to guide future quince breeding and management studies.

Pressure-independent cerebrovascular remodelling and changes in myogenic reactivity in diabetic Goto-Kakizaki rat in response to glycaemic control.

AIM: We have shown hypertrophic cerebrovascular remodelling in the Goto-Kakizaki (GK) rat model of diabetes. This study tested the hypotheses that (1) vascular remodelling develops as the disease progresses and alters myogenic reactivity of resistance vessels important for regulation of cerebral blood flow (CBF), and (2) glycaemic control prevents cerebrovascular remodelling and myogenic dysfunction. METHODS: Middle cerebral artery (MCA) lumen diameter, media : lumen (M : L) ratio, cross-sectional area (CSA) and myogenic tone were measured in 10- and 18-week-old control Wistar and diabetic GK rats using pressurized arteriograph (n = 8-14/group). Mean arterial blood pressure (MAP) was measured with telemetry (n = 5/group). Additional GK rats were treated with metformin (300 mg kg(-1) day(-1) ) for glycaemic control starting at 7 weeks after the onset of diabetes until 18 weeks (n = 9). RESULTS: In the control group, there was no difference in remodelling indices, myogenic tone or MAP between ages. Eighteen week diabetic rats displayed increased M : L ratio and CSA, but decreased lumen diameter and myogenic tone compared to 10-week GK or 18-week control rats. MAP increased starting around 10 weeks of age and remained slightly higher in the GK rats. Glycaemic control normalized M : L ratio, CSA, lumen diameter and myogenic tone with no effect on blood pressure. CONCLUSIONS: These findings suggest that diabetic rats develop MCA remodelling as the disease progresses but this is associated with impaired myogenic reactivity which may ultimately affect CBF. Our results also provide evidence that glycaemic control is an effective therapeutic strategy to prevent cerebrovascular remodelling and dysfunction.

Amplified fragment length polymorphism analysis of grapevine rootstock genotypes in Turkey.

Amplified fragment length polymorphism (AFLP) technique was used to assess genetic relationships among 20 grapevine rootstocks in Turkey. Discrimination of the rootstocks with 10 primer combinations yielded 1366 bands on AFLP gels; 65% of them were polymorphic. The rootstocks revealed two main clusters; one of them comprised two (Malégue and Harmony) and the other 18 genotypes. The Ber x Rip hybrids Cosmo 2 and Cosmo 10 formed a group with a high internal similarity ratio (0.909); they also formed a group with other Ber x Rip hybrids, 5C, 8B, SO4, and 420A Mgt, with a similarity ratio higher than 0.690 (subcluster II). Rootstock 5BB was placed in another subcluster (subcluster III). Among five Ber x Rup rootstocks, 110R-99R (0.853) and 1103P-140Ru (0.837), which were located in different subclusters, formed a dual group, as expected. Rootstock 779P, which had almost 0.800 similarity with the dual group of 110R-99R, formed another group. The 44-53 Malégue and Harmony rootstocks formed a group with the lowest similarity ratio (0.668) (subcluster I) and 41B-Fercal formed another dual group with a high similarity ratio (0.813). The distinction capacity of single- and double-EcoRI-MseI primers was evaluated; primers AC/CTA, TC/CAC, AG/CTC, and AG/CAG discriminated the 20 rootstocks, with a similarity value below 0.910. The best primers for discrimination of rootstock varieties were AG/CAG and AG/CTC, while the primers TC/CAC and AC/CTA could also be useful for clonal discrimination of genotypes.

SSR-based molecular analysis of economically important Turkish apricot cultivars.

Turkey is not only the main apricot (Prunus armeniaca) producer and exporter in the world, but it also has a wide variety of apricot germplasms, owing to its close proximity to the centers of apricot origin. However, there is little or no genetic information on many apricot cultivars that are extensively cultivated in Turkey. We examined the genetic relatedness of 25 Turkish and four exotic apricot cultivars using SSR (simple sequence repeat) markers that were either previously developed for apricot, or for peach (P. persica), a close relative of apricot. Allele diversity (with an average allele number of 6.37) at the SSR loci and the heterozygosity rates (with an average Ho value of 0.648) of these cultivars were found to be higher than in previous studies that used the same loci for apricot. This fact might be attributed to the analysis of different numbers of accessions in the different studies. No correlations were found between the genetic relatedness and the geographical distributions of these cultivars. The data reported here will assist in the prevention of confusions in the apricot propagation and breeding in Turkey. The findings can also be directly compared with other studies that used the same SSR markers on apricot.

Genetic characterization of green bean (Phaseolus vulgaris) genotypes from eastern Turkey.

Green bean genotypes collected from eastern Turkey were characterized using simple sequence repeat (SSR) markers and morphological traits. Among 12 SSR markers, 10 produced successful amplifications and revealed DNA polymorphisms that were subsequently used to assess genetic relatedness of the genotypes. Based on the number of alleles generated and the probability of identity values, the most informative SSR loci were PVGLND5, PVMEIG, PV-ag001, and PV-ag004. Probably, due to the inbreeding nature of beans, the heterozygosity observed within genotypes was low at most of the SSR loci. The UPGMA dendrogram constructed based on the SSR data yielded two major clusters. The overall genetic distance was around 98%, among the genotypes. This information can be used to help select Turkish green bean lines.

Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

Potential role of endothelin receptor antagonists in the setting of cardiopulmonary bypass: relevance to myocardial performance.

The ET system is activated in cardiac surgical setting as evidenced by elevated systemic and myocardial ET-1 levels after coronary bypass grafting surgery which requires hypothermic cardioplegic arrest and cardiopulmonary bypass. Increased ET-1 may influence a number of clinical parameters in this setting. First, ET-1 may directly modulate myocardial contractile performance in the early postoperative period resulting in LV dysfunction and a complex postoperative course. Second, elevated ET-1 levels may exacerbate increased pulmonary vascular resistance and contribute to the development of transient pulmonary hypertension following bypass. Finally, augmented postoperative ET-1 levels could contribute to changes in the caliber and flow of vascular conduits used for coronary bypass. In this review, a current perspective on the ET system in the setting of cardiopulmonary bypass grafting surgery is provided and the potential use of ET receptor antagonists in this setting is discussed.

beta-Adrenergic and endothelin receptor interaction in dilated human cardiomyopathic myocardium.

BACKGROUND: Although end-stage dilated cardiomyopathy (DCM) is characterized by defects in beta-adrenergic receptor (beta-AR) activity and increased endothelin-1 (ET-1), possible interactions between these 2 systems remain to be defined. Accordingly, the goal of this study was to determine the effects of ET receptor activation on beta-AR signaling through measurement of cyclic adenosine monophosphate (cAMP) in normal and DCM myocardium. METHODS AND RESULTS: Myocardial sarcolemmal preparations were prepared from normal human (n = 6), dilated cardiomyopathic (n = 10), and ischemic cardiomyopathic (ICM, n = 10) tissue. Basal cAMP production was measured in the presence of ET-1 alone (10(-6) to 0(-9) mol/L) as well as after (-)isoproterenol (10(-6) to 10(-2) mol/L) or forskolin (0.05 to 30.0 micromol/L) stimulation. beta-AR and ET receptor profiles were determined by radiolabeled ligand assays. ET-1 inhibited basal cAMP production in all preparations in a concentration-dependent manner. However, beta-AR-stimulated cAMP production by either isoproterenol or forskolin was not significantly affected by ET-1. beta-AR receptor density was reduced, and a selective reduction of the ET(B) receptor occurred in both forms of DCM. CONCLUSIONS: Under basal conditions, ET receptor stimulation reduced cAMP levels, which may influence contractility, particularly with DCM.

Expression and activity of pulmonary endothelin converting enzyme in heart failure: relation to endothelin biosynthesis and receptor distribution.

BACKGROUND: Although reduced pulmonary clearance of endothelin-1 (ET-1) has been suggested to contribute to increased circulating levels in congestive heart failure (CHF), the regulation of the pulmonary ET system with CHF remains to be defined. Accordingly, the aim of the present study is to investigate the expression and activity of the ET system with the development of CHF. METHODS AND RESULTS: Pulmonary tissue samples were collected from pigs with pacing CHF (240 bpm, 3 wks, n = 10) and controls (n = 10). The pulmonary messenger RNA (mRNA) and protein levels of endothelin converting enzyme-1 (ECE-1) subisoforms, ET-1, and ET receptor profiles were determined. The gene expression of ET-1 precursor, ECE-1a, and ET(A) was upregulated 4-, 3-, and 2-fold, respectively, with CHF. Pulmonary tissue ET-1 was increased to 13 +/- 2 fmol/mg protein from control values of 5 +/- 1 fmol/mg protein (P <.05), and ECE-1 activity was augmented from 3,264 +/- 665 fmol/mg protein in control animals to 14,073 +/- 654 fmol/mg protein per hour in CHF animals (P <.05). The ET(B) receptor density decreased, whereas ET(A) receptors were increased in CHF, indicating a shift in the ET(A) to ET(B) ratio. CONCLUSIONS: Both the increased synthesis and the decreased clearance of ET-1 via ET(B) receptors may contribute to the increased systemic and pulmonary ET-1 levels in CHF.

A review of endothelin and hypertension in African-American individuals.

Endothelin-1 (ET-1) is a peptide with potent vasopressor and mitogenic actions. Moreover, ET-1 displays modulatory effects on the endocrine system, including stimulation of angiotensin II and aldosterone production, and influences ion and fluid transport in the gut and kidney. A number of groups reported that ET-1 is overexpressed in the vasculature in several salt-sensitive models of experimental hypertension. African Americans present with a salt-sensitive and low-renin model of hypertension, and circulating plasma ET-1 levels are significantly increased in this population. The prevalence of hypertension and its complications is also higher in Blacks than in Whites and, despite extensive research, the reasons for this difference are not well understood. We propose that vasoactive, mitogenic, and renal effects of the ET system might contribute to the development, maintenance and/or complications of hypertension in African Americans.

Role of the hydrophobic transmembrane domain in membrane anchoring of endothelin-converting enzyme-1a.

Endothelin-converting enzyme-1 (ECE-1) is a type II membrane protein that cleaves big endothelin-1 (big ET-1) to endothelin-1 (ET-1). The role of the N-terminal and membrane-spanning signal anchor domains in the biosynthesis and function of ECE-1 isoforms, ECE-1a, ECE-1b and ECE-1c, remains unknown. This study provides evidence that the deletion of the cytoplasmic N-terminal tail (residues 1-55) of bovine ECE-1a results in the processing of a putative signal peptide located in the signal anchor domain leading to the partial secretion of the recombinant enzyme into the media. The truncation of N-terminal and/or signal anchor domain does not affect the activity of ECE-1a. These results indicate that the hydrophobic signal anchor domain alone is not sufficient for the membrane anchoring of ECE-1a and that the N-terminal domain of ECE-1a is important for membrane targeting as well as the intracellular localization of the enzyme.

Temporal endothelin dynamics of the myocardial interstitium and systemic circulation in cardiopulmonary bypass.

OBJECTIVE: Increased systemic levels of the bioactive peptide endothelin 1 during and after cardioplegic arrest and cardiopulmonary bypass have been well documented. However, endothelin 1 is synthesized locally, and therefore myocardial endothelin 1 production during and after cardiopulmonary bypass remains unknown. METHODS: Pigs (n = 11) were instrumented for cardiopulmonary bypass, and cardioplegic arrest was initiated. Myocardial interstitial and systemic arterial levels of endothelin 1 were measured before cardiopulmonary bypass, throughout bypass and cardioplegic arrest (90 minutes), and up to 90 minutes after separation from bypass. Myocardial interstitial endothelin 1 was determined by microdialysis and radioimmunoassay. RESULTS: Baseline myocardial endothelin 1 levels were higher than systemic endothelin 1 levels (25.6 +/- 6.7 vs 8.3 +/- 1.1 fmol/mL, P <.05). With the onset of bypass, myocardial endothelin 1 increased by 327% +/- 92% from baseline (P <.05), which preceded the increase in systemic endothelin 1 levels. CONCLUSION: Myocardial compartmentalization of endothelin 1 exists in vivo. Cardiopulmonary bypass and cardioplegic arrest induce temporal differences in endothelin 1 levels within the myocardial interstitium and systemic circulation, which, in turn, may influence left ventricular function in the postbypass period.

Secretion of endothelin converting enzyme-1a: the hydrophobic signal anchor domain alone is not sufficient to promote membrane localization.

Endothelin converting enzyme-1 (ECE-1) is a type II membrane protein that is important for the proteolytic activation of big endothelin-1 to endothelin-1. Although the highly conserved zinc-binding motif is known to be located in the extracellular domain, the role(s) of the N-terminal and membrane-spanning signal anchor domains in the biosynthesis and function of ECE-1 isoforms, ECE-1a, ECE-1b, and ECE-1c, remain undetermined. In this study, we provide evidence that the deletion of the cytoplasmic N-terminal tail (residues 1-55) of ECE-1a results in the cleavage of a potential signal peptide located in the signal anchor domain leading to the partial secretion of the recombinant enzyme into the media. However, the truncation of N-terminal and/or signal anchor domain does not affect the activity of ECE-1a. Therefore, our results demonstrate that the hydrophobic signal anchor domain alone is not sufficient for the membrane anchoring of ECE-1a and that the N-terminal domain of ECE-1a is important for membrane targeting as well as the intracellular localization of the enzyme.