Shahida Shafi

Heat shock protein 27: its potential role in vascular disease.

Heat shock proteins are molecular chaperones that have an ability to protect proteins from damage induced by environmental factors such as free radicals, heat, ischaemia and toxins, allowing denatured proteins to adopt their native configuration. Heat shock protein‐27 (Hsp27) is a member of the small Hsp (sHsp) family of proteins, and has a molecular weight of approximately 27 KDa. In addition to its role as a chaperone, it has also been reported to have many additional functions. These include effects on the apoptotic pathway, cell movement and embryogenesis. In this review, we have focused on its possible role in vascular disease.

Anti-heat shock protein-27 (Hsp-27) antibody levels in patients with chest pain : Association with established cardiovascular risk factors

BACKGROUND Antibody titres to several heat shock proteins (Hsps) have been shown to be associated with risk factors for cardiovascular disease (CVD), but there are no data for Hsp-27. We developed an ELISA for total IgG antibody concentrations, applying this to individuals with and without acute coronary syndrome, and have assessed the relationship between antibody levels and individual coronary risk factors. METHODS Blood was collected from 63 healthy controls without a history of chest pain or CVD and 60 patients admitted to hospital with acute cardiac chest pain on admission and approximately 12 h after the acute event. RESULTS Patients with chest pain had significantly higher Hsp-27 antibody levels than controls [median 0.16 (range 0.01-0.51) vs. 0.10 (range 0.00-0.32); p<0.001]. Furthermore, Hsp-27 antibody concentrations showed strong associations with age and hypertension (Standardised beta coefficient=0.343, p<0.001 and = -0.235, p<0.016, respectively), but not with other established cardiovascular risk factors. Logistic regression analysis showed age and diabetes were significant predictors of risk of CVD with OR 1.29 (95% CI 1.16 to 1.42, p=0.001) and 25.9 (95% CI 2.14>312, p=0.01) respectively. CONCLUSIONS Raised antibody levels to Hsp-27 were associated only with age and hypertension.

Serum insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation status in subjects with and without ischaemic heart disease

BACKGROUND Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the activity of IGF-I. It exists in serum as phosphorylated and less phosphorylated forms. We wished to measure serum levels of both these forms of IGFBP-1, using a novel assay, in subjects with, or without ischaemic heart disease (IHD). METHODS We measured serum concentrations of the phosphorylated and less phosphorylated forms of IGFBP-1 in 75 subjects (36 with and 39 without IHD). Two immunoassays were used, one which detects non-, and less-phosphorylated forms (LpIGFBP-1), and another which specifically detects the serine phosphorylated form of IGFBP-1 (pIGFBP-1). RESULTS LpIGFBP-1 concentrations were significantly higher in subjects without IHD than in those with IHD (5.3+/-0.5 microg/L vs. 2.7+/-0.4 microg/L, p<0.001). pIGFBP-1 levels were also significantly higher in subjects without IHD compared to those with IHD (33.3+/-2.0 microg/L vs. 25.3+/-2.2 microg/L, p<0.01). The correlation between LpIGFBP-1 and pIGFBP-1 for all subjects was (r=0.71, p<0.001). This association was stronger in subjects without IHD (r=0.76, p<0.001) than for those with IHD (r=0.60, p<0.001). A significant negative association was observed between IGF-I and the ratio between the two forms (r=-0.45, p<0.0001). Receiver-Operating Characteristic (ROC) curve showed the highest area under the curve for LpIGFBP-1 (0.75) [95% CI: 0.63-0.86] and optimum cut-off value of 2.83 microg/L with 75% sensitivity and 74% specificity. CONCLUSIONS We propose that low serum concentrations of IGFBP-1 forms could be a marker of coronary risk, and the LpIGFBP-1:pIGFBP-1 ratio may be an index of biologically active IGF-I.

Periadventitial delivery of anti-EGF receptor antibody inhibits neointimal macrophage accumulation after angioplasty in a hypercholesterolaemic rabbit

Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR‐specific, blocking monoclonal antibody (ICR62, which inhibits EGF‐binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol‐diet. Two weeks after the initiation of the diet, a balloon‐catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype‐matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB‐EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon‐catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR‐binding such as ICR62.

Effects of reserpine on expression of the LDL receptor in liver and on plasma and tissue lipids, low density lipoprotein and fibrinogen in rabbits in vivo

The effects of administering reserpine (0.1 mg/kg) or 17alpha-ethinyloestradiol (2.5 mg/kg) to New Zealand White rabbits on low density lipoprotein receptors in liver, on plasma low density lipoprotein and fibrinogen and on plasma and tissue lipids were determined. Blood pressure and heart rate were also followed. The drugs were injected subcutaneously into conscious unrestrained rabbits for 5 days. On the 6th day homologous 125I-tyramine cellobiose labelled low density lipoprotein (125I-TC-LDL) was injected intravenously and 24 h later the animals were killed. Compared to controls, reserpine significantly increased LDL receptor expression in the liver by about threefold, and reduced total cholesterol in plasma, aorta and heart, without affecting plasma triglycerides. The reductions in plasma cholesterol and heart were due to decreases in both unesterified and esterified cholesterol. Similar effects were observed with oestrogen, except that there was no change in esterified cholesterol in aorta. In liver, a decrease of 24% in total cholesterol was due mainly to decreased esterified cholesterol. In adrenal glands total cholesterol increased by 25%. Reserpine significantly accelerated the plasma clearance of intravenously injected homologous 125I-TC-LDL and reduced its accumulation in aortic wall. Neither reserpine nor oestradiol affected blood pressure, haematocrit or plasma fibrinogen. The results suggest that reserpine is an affective anti-atherogenic drug capable of decreasing cholesterol in plasma, arteries and heart by increasing high affinity LDL receptors in the liver.

Insulin sensitivity (Si) assessment in lean and overweight subjects using two different protocols and updated software

Abstract Background. The standard frequently-sampled intravenous glucose tolerance test (FSIVGTT) is an alternative procedure to the clamp technique for estimating the insulin sensitivity (Si) parameter. The goal of this study was to compare Si in lean and overweight individuals in addition to assessing intra-individual reproducibility using two different protocols and updated software. Methods. FSIVGTT was carried out in 14 lean (BMI ≤ 25 kg/m2) and 14 overweight (BMI>25 kg/m2) subjects using two different protocols; full (29 samples) and short (12 samples). For reproducibility assessment four normal subjects (triplicate on three and twice on one) were recruited to undergo the same procedure at 1-week intervals. Data analysis was performed using COMAL and Minmod Millennium software. Results. Mean Si (10−4min−1[pmol/l]−1) values were significantly different between lean and overweight subjects (p < 0.001) but not between the two protocols using both software packages. For the full and short protocols, Si values were more closely related in lean versus overweight subjects using either COMAL (r = 0.98, p < 0.001), (r = 0.89, p < 0.001) or Minmod Millennium (r = 0.99, p < 0.001), (r = 0.85, p < 0.001) software respectively. The intra-individual reproducibility (%CV) of Si (COMAL) in full versus short protocol was 18.3 ± 11.1% and 13.7 ± 1.9% respectively. Reproducibility values for Si (Minmod Millenium) in full versus short protocols were 14.3 ± 3.8 and 14.9 ± 1.9% respectively. Conclusions. Si can be assessed accurately by a short protocol FSIVGTT in normal individuals. The short protocol may give less acceptable results for insulin sensitivity in individuals who have normal glucose tolerance but high BMI.

Long-term low-dose treatment with reserpine of cholesterol-fed rabbits reduces cholesterol in plasma, non-high density lipoproteins and arterial walls

The effects of long-term low-dose treatment with reserpine on plasma lipoproteins and arterial cholesterol were determined in cholesterol-fed rabbits. Hepatic low-density lipoprotein (LDL) receptors; uptake of LDL by liver, heart, and kidneys; plasma fibrinogen; blood pressure; and heart rate were also determined. Reserpine at 43 &mgr;g/kg · d was continuously infused subcutaneously via implanted minipumps for 6 weeks into conscious unrestrained male New Zealand White rabbits (n = 5) fed a 0.2% cholesterol-enriched diet. Compared with controls, reserpine (n = 4) significantly reduced the elevated levels of plasma total cholesterol and esterified and unesterified cholesterol throughout the study, and at 6 weeks of treatment these reductions were 42, 41, and 49%, respectively. The increased cholesterol in the aortic walls (n = 5) produced by the atherogenic diet was reduced by 73% (p < 0.004) and 125I-tyramine cellobiose-labeled LDL by 67 to 86% (0.05 < p <0.004), respectively. The aortic intimal-medial thickness ratio was reduced by 70%. The decrease in elevated plasma total cholesterol was mainly due to cholesterol reductions in both LDL (41%) and non–high density lipoprotein (HDL) of density < 1.019 g/ml (51%). HDL cholesterol and triglyceride levels were unchanged. Reserpine had no significant effects on the clearance of 125I-tyramine cellobiose-LDL from plasma and there was a trend towards an increase in hepatic LDL receptor expression. Heart rate was decreased by 28%. There were no significant effects on blood pressure, liver and heart lipids, hematocrit, or plasma fibrinogen. The results suggest that treatment of cholesterol-fed rabbits with reserpine at a low dose over a long period prevents increases in plasma atherogenic lipoproteins. Reserpine decreases the cholesterol in aortic walls and the intima-media thickness ratio. This anti-atherosclerotic effect of reserpine may have therapeutic implication.

Increased expression of phosphorylated forms of heat-shock protein-27 and p38MAPK in macrophage-rich regions of fibro-fatty atherosclerotic lesions in the rabbit

We aimed to assess the expression and distribution of Hsp27, pHsp27 (Ser82), p38MAPK and p‐p38MAPK in fibro‐fatty atherosclerotic lesions and the myocardium of hypercholesterolaemic rabbits. Male New Zealand white rabbits were fed a high‐cholesterol diet for 18 weeks, maintaining serum cholesterol at approximately 20 mmol/l over this period. Aortic arch and myocardial tissues were analysed by Western blot, immunohistochemistry and double immunofluorescence. Plasma Hsp27 levels were measured by ELISA. There was a significant increase in the expression of monomeric and dimeric forms of Hsp27, together with pHsp27 (Ser82), p38MAPK and p‐p38MAPK in the fibro‐fatty atherosclerotic lesions (P < 0.01; P < 0.05; P < 0.001; and P < 0.001, respectively) and the myocardial tissues (P < 0.001) from the cholesterol‐fed rabbits compared with equivalent tissues from controls when the plasma concentration was low. Immunohistochemical analysis of the fibro‐fatty lesions showed marked increases in Hsp27 and pHsp27 (Ser82) immunoreactivity. Double immunostaining showed intense expression of pHsp27 and p‐p38MAPK in regions that were rich in macrophages, suggesting a close association with these inflammatory cells, whereas, in regions rich in smooth muscle cells, only p‐p38MAPK was found to be strongly expressed. An increased expression of pHsp27 (Ser82) was spatially associated with increased p‐p38MAPK within fibro‐fatty atherosclerotic lesions and was colocalized to regions rich in macrophages. The initial increase in plasma Hsp27 levels may reflect the increase in systemic inflammation and oxidative stress in the early phases of disease. The falling concentrations subsequently may be coincident with the development of the advanced atherosclerotic lesions.

Serum intercellular adhesion molecule-1 (ICAM-1) and high-sensitivity (hs) C-reactive protein (CRP) in adolescent females: physical activity and dietary influences

CRP is an inflammatory marker that has been shown to be affected by dietary factors. Recent investigations suggest that physical activity (PA) may be associated with reduced serum CRP levels (1) . Intercellular adhesion molecule-1 (ICAM-1) is a trans-membrane glycoprotein that plays a key role in leucocyte migration and activation. ICAM-1 is up regulated on activated endothelial cells and its soluble form (sICAM-1) is increased in athletes (2) . Intense physical training (3) and dietary factors (4) may also effect its serum concentrations, although confounding effects of diet and adiposity have not been taken into account. Furthermore, many of these studies have been conducted in adults, in whom the onset of atherosclerosis may be a further confounding issue. The aim was to assess whether serum sICAM-1 and hsCRP differ between physically-active female gymnasts and inactive females who were part of a 3-year longitudinal bone investigation (5) . A further aim was to assess the association between these two variables and BMI, dietary intake and PA. Healthy girls, including twenty-five competitive gymnasts and nineteen healthy sedentary individuals aged 8‐17 years (13.00.7 years; 12.00.5 years) respectively, were recruited. Human sICAM-1 and hsCRP were measured using commercial ELISA. Serum sICAM (mg/L) was significantly higher in the gymnasts compared with the sedentary girls (0.23 (SE 0.01) v. 0.29 (SE 0.015); P < 0.003), whilst serum hsCRP concentrations (mg/L) were significantly lower (0.49 (SE 0.03) v. 1.38 (SE 0.19); P < 0.0003). These differences remained significant after adjustment for body weight and height (analysis of covariance; P < 0.005). Weight, height, BMI and dietary factors were all significantly lower in gymnasts compared with controls, whilst PA was significantly higher in gymnasts (P < 0.05 for all variables). Among the gymnasts serum sICAM was inversely related to dietary total fat, MUFA and SFA (Table), but not to any anthropometric measures or levels of PA (by univariate analysis). Serum hsCRP was not significantly related to any of the variables.