Beta Blockade Prevents Cardiac Morphological and Molecular Remodelling in Experimental Uremia.

Heart failure and chronic kidney disease (CKD) share several mediators of cardiac pathological remodelling. Akin to heart failure, this remodelling sets in motion a vicious cycle of progressive pathological hypertrophy and myocardial dysfunction in CKD. Several decades of heart failure research have shown that beta blockade is a powerful tool in preventing cardiac remodelling and breaking this vicious cycle. This phenomenon remains hitherto untested in CKD. Therefore, we set out to test the hypothesis that beta blockade prevents cardiac pathological remodelling in experimental uremia. Wistar rats had subtotal nephrectomy or sham surgery and were followed up for 10 weeks. The animals were randomly allocated to the beta blocker metoprolol (10 mg/kg/day) or vehicle. In vivo and in vitro cardiac assessments were performed. Cardiac tissue was extracted, and protein expression was quantified using immunoblotting. Histological analyses were performed to quantify myocardial fibrosis. Beta blockade attenuated cardiac pathological remodelling in nephrectomised animals. The echocardiographic left ventricular mass and the heart weight to tibial length ratio were significantly lower in nephrectomised animals treated with metoprolol. Furthermore, beta blockade attenuated myocardial fibrosis associated with subtotal nephrectomy. In addition, the Ca++- calmodulin-dependent kinase II (CAMKII) pathway was shown to be activated in uremia and attenuated by beta blockade, offering a potential mechanism of action. In conclusion, beta blockade attenuated hypertrophic signalling pathways and ameliorated cardiac pathological remodelling in experimental uremia. The study provides a strong scientific rationale for repurposing beta blockers, a tried and tested treatment in heart failure, for the benefit of patients with CKD.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan.

Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI=±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI=±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P≤0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI=±7.86) and 11 (5.6%, 95% CI=±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI=±6.58) and 23 (18.8%, 95% CI=±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI=±5.62) and 32 (11.3%, 95% CI=±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P≤0.05) between all age groups. Statistically, no significant (P≥0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI=±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI=±10.4) and crossbred (19 of 239, 8.3%, 95% CI=±7.47), indicating a significant (P≤0.05) trend of positivity between various breeds of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen.

Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple test and by antigen ELISA, respectively. We accept our hypothesis based on these findings. We also demonstrated that fecal samples collected from the youngest age group (1 mo-5 yr) were more likely to be positive for E. histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

Interleukin-1 has opposing effects on connective tissue growth factor and tenascin-C expression in human cardiac fibroblasts.

Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10ng/ml, 6h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1-3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.

Novel G protein-coupled oestrogen receptor GPR30 shows changes in mRNA expression in the rat brain over the oestrous cycle.

Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and ß in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM) and periaqueductal gray (PAG) GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX) rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI) colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

MMP-3 (5A/6A) polymorphism does not influence human smooth muscle cell invasion.

BACKGROUND: Stromelysin (MMP-3) is an important regulator of vascular smooth muscle cell (SMC) invasion, a key contributor to saphenous vein (SV) bypass graft failure. The 5A allele of the common -1612 MMP-3 5A/6A promoter polymorphism reportedly confers increased promoter activity, MMP-3 tissue expression, and susceptibility to a number of vascular pathologies. The aim of this study was to determine whether the MMP-3 5A/6A polymorphism directly influences endogenous MMP-3 expression levels and, consequently, cell invasion, in SV-derived SMC cultured from patients with different genotypes. MATERIAL AND METHODS: Genotyping of 226 patients revealed -1612 MMP-3 5A/6A genotype frequencies of 20.8% 5A/5A, 52.7% 5A/6A, and 26.5% 6A/6A. Using a standardized, controlled protocol, we investigated cytokine- and growth factor-induced MMP-3 expression (real-time polymerase chain reaction [RT-PCR], ELISA) and SV-SMC invasion (Boyden chamber with Matrigel barrier) using cultured SV-SMC from patients with different MMP-3 genotypes. RESULTS: Despite observing a strong correlation between MMP-3 mRNA levels and MMP-3 protein secretion, no significant differences were apparent in MMP-3 expression levels or cell invasion between cells with different MMP-3 5A/6A genotypes. CONCLUSIONS: Our data suggest that the MMP-3 5A/6A promoter polymorphism in isolation does not influence levels of MMP-3 secretion or cellular invasion in human SV-SMC.

Morphological and molecular characterisation of Echinococcus granulosus in livestock and humans in Punjab, Pakistan.

Cystic echinococcus poses an important economic and public health problem to Pakistan. Our study determined the prevalence and genotypes of Echinococcus present in domestic livestock and humans in Punjab, Pakistan. Out of 39,738 animals examined, 6.67% of animals were found infected. The prevalence and fertility of hydatid cysts was highest in camels (prevalence 17.29%; proportion fertile 95%), followed by sheep (prevalence 7.52%; proportion fertile 86.4%), buffalo (prevalence 7.19%; proportion fertile 84.3%), goats (prevalence 5.48%; proportion fertile 79.09%) and cattle (prevalence 5.18%; proportion fertile 75.25%). Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the common sheep strain (G1) and buffalo strain (G3) are cycling among livestock in Punjab and that these strains are highly adapted to goats, camels and cattle. Both human cysts were found to belong to the common sheep strain (G1) of E. granulosus, reinforcing this strain has the most potential for zoonotic transfer. Both morphological and molecular results support earlier studies suggesting that Echinococcus of sheep and buffalo origin is phenotypically and genetically similar which adds further evidence to support its recognition as one species viz, Echinococcus granulosus sensu stricto.

The -1562C/T MMP-9 promoter polymorphism does not predict MMP-9 expression levels or invasive capacity in saphenous vein smooth muscle cells cultured from different patients.

Matrix metalloproteinase-9 (MMP-9) is an important regulator of vascular smooth muscle cell (SMC) invasion and proliferation. The T allele of the -1562C/T MMP-9 promoter polymorphism reportedly confers increased MMP-9 promoter activity, plasma MMP-9 levels and susceptibility to vascular pathologies. The aim of this study was to determine whether the MMP-9 -1562C/T polymorphism directly influences endogenous MMP-9 expression levels in saphenous vein (SV) SMC cultured from patients with different genotypes. Genotyping of 408 patients revealed -1562C/T genotype frequencies of 73.3% CC, 25.0% CT and 1.7% TT. Using a standardized, controlled protocol we investigated the effects of phorbol ester (TPA) and a physiological stimulus (PDGF+IL-1) on MMP-9 expression in cultured SV-SMC from 15 CC, 15 CT and 3 TT patients, and on PDGF+IL-1-induced SV-SMC invasion (Boyden chamber with Matrigel barrier). A strong correlation between MMP-9 mRNA levels (real-time RT-PCR) and MMP-9 protein secretion (gelatin zymography) was observed. However, no significant differences were observed in MMP-9 expression levels, or in SV-SMC invasion, between cells with different -1562C/T genotypes. Moreover, MMP-9 promoter activity of the C and T variants was similar. Our data challenge the functional nature of the -1562C/T polymorphism and its capacity to modulate MMP-9 expression levels and SV-SMC invasion, and hence susceptibility to vascular pathologies in vivo.

Oestrogen receptors in the central nervous system and evidence for their role in the control of cardiovascular function.

Oestrogen is considered beneficial to cardiovascular health through protective effects not only on the heart and vasculature, but also on the autonomic nervous system via actions on oestrogen receptors. A plethora of evidence supports a role for the hormone within the central nervous system in modulating the pathways regulating cardiovascular function. A complex interaction of several brainstem, spinal and forebrain nuclei is required to receive, integrate and co-ordinate inputs that contribute appropriate autonomic reflex responses to changes in blood pressure and other cardiovascular parameters. Central effects of oestrogen and oestrogen receptors have already been demonstrated in many of these areas. In addition to the classical nuclear oestrogen receptors (ERalpha and ERbeta) a recently discovered G-protein coupled receptor, GPR30, has been shown to be a novel mediator of oestrogenic action. Many anatomical and molecular studies have described a considerable overlap in the regional expression of these receptors; however, the receptors do exhibit specific characteristics and subtype specific expression is found in many autonomic brain areas, for example ERbeta appears to predominate in the hypothalamic paraventricular nucleus, whilst ERalpha is important in the nucleus of the solitary tract. This review provides an overview of the available information on the localisation of oestrogen receptor subtypes and their multitude of possible modulatory actions in different groups of neurochemically and functionally defined neurones in autonomic-related areas of the brain.

The clinical significance of a common, functional, X-linked angiotensin II type 2-receptor gene polymorphism (-1332 G/A) in a cohort of 509 families with premature coronary artery disease.

AIMS: To assess, in families with premature coronary artery disease (CAD), the possible association, with linkage, of the X-linked AT2 receptor (-1332 G/A) gene polymorphism and premature CAD. METHODS AND RESULTS: We investigated 509 families with a history of premature CAD that consisted of one sibling affected with premature CAD and two unaffected siblings. Genotyping of subjects was performed using a restriction enzyme digestion of an initial 310 bp polymerase chain reaction fragment that included the AT2 (-1332 G/A) locus. The mean age of the 611 individuals affected by premature CAD at the time of event was 49.5 +/- 8.1 years. Conditional logistic regression analysis confirmed a significant predictive value of premature CAD for the covariates of hypertension, diabetes, dyslipidaemia, history of smoking, and male gender. The genetic data were analysed for these families using the X-linked sibling transmission/deletion test (XS-TDT) statistics program. In hemizygous men we observed evidence for association in the presence of linkage, for the AT2 (-1332 G/A) locus and premature CAD (P-exact value = 0.024) and also a trend towards association, in the presence of linkage, for this polymorphism and hypertension (P-exact value = 0.08). CONCLUSIONS: We have observed evidence of association between the presence of linkage for the X-linked AT2 (-1332 G/A) polymorphism and premature CAD in hemizygous males.