Sex steroids modulate human aortic smooth muscle cell matrix protein deposition and matrix metalloproteinase expression

Large artery stiffening increases cardiovascular risk and promotes isolated systolic hypertension which is more prevalent in elderly women than men. Variation in sex steroid levels between males and females and throughout life may modulate arterial stiffness. We hypothesized that sex steroids directly influence expression of important structural proteins which determine arterial biomechanical properties. Human aortic smooth muscle cells were incubated with physiological concentrations of 17beta-estradiol, progesterone, 17beta-estradiol and progesterone, or testosterone for 4 weeks. Collagen, elastin, and fibrillin-1 deposition was examined (histochemistry/immunohistochemistry). Gene and protein expression of 2 important matrix metalloproteinases (MMPs), MMPs 2 and 3, regulating matrix turnover was assessed. All sex steroids reduced collagen deposition relative to control (100%). However, the reduction was greater with female sex steroids than testosterone (control, 100%; 17beta-estradiol plus progesterone, 20+/-2%; testosterone 74+/-12%, P<0.001). Female sex steroids increased elastin deposition compared with control (control, 100%; 17beta-estradiol, 540+/-60%; progesterone, 290+/-40%; 17beta-estradiol plus progesterone, 400+/-80%, all P<0.01). The elastin/collagen ratio was >11-fold higher in the presence of 17beta-estradiol and progesterone compared with testosterone. Fibrillin-1 deposition was doubled in the presence of female sex steroids (17beta-estradiol plus progesterone) compared with testosterone (P<0.01). MMP-2 gene and protein expression was unaffected by any sex steroid. Testosterone increased both gene and protein expression of MMP-3 relative to both control and female sex steroids (P<0.01). This may contribute to degradation of elastic matrix proteins. In conclusion, female sex steroids promote an elastic matrix profile, which likely contributes to variation in large artery stiffness observed between sexes and with changes in hormonal status across the lifespan.     

Elevated matrix metalloproteinase expression after stent implantation is associated with restenosis

OBJECTIVE: Matrix metalloproteinases (MMPs) play a key role in intimal growth and is responsible for ventricular remodeling after stent implantation. However, little is known about the relationship between early MMPs expression post-stent implantation and follow-up restenosis. METHODS: We investigated the serial changes of serum MMP-9, MMP-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in 16 control subjects with normal coronary angiography (control) and 40 patients before and on the 1st, 3rd and 7th day after uncomplicated stent implantation. Follow-up angiography was performed at 6 months after stent implantation. RESULTS: Serum MMP-2 level was higher in patients with restenosis on the 1st day post-stent implantation and returned to pre-operation level thereafter. Serum MMP-9 levels consistently increased in patients with restenosis up to 7th day post-stent implantation; MMP-9 levels in the 1st, 3rd and 7th day after stent implantation were positively correlated to the late loss index 6 months after stent implantation. CONCLUSIONS: Increased serum MMP-9 level is associated with increased risk of restenosis post-stent implantation.     

Genetic selection for insulin-like growth factor-1 in growing mice is associated with altered growth

Substantial responses in the 6-week and mature body-weights of mice occurred after 7 generations of selection for or against plasma levels of Insulin-like Growth Factor-1 (IGF-1). Plasma levels of IGF-1 were also significantly different after 7 generations of selection (high line = 85 +/- 2 ng/ml, low line = 58 +/- 2 ng/ml). The average 6-week weight in the line selected for high plasma IGF-1 was 22.5 +/- .2 g compared with 18.5 +/- .2 g in the low plasma IGF-1 line, after 7 generations of selection. The difference between lines was maintained at 20 weeks of age. These data provide further evidence for the roles of IGF-1 in the regulation of somatic growth and as a mediator of a genetic component of growth.     

Downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is associated with enhanced expression of matrix metalloproteinases and cholangiocarcinoma metastases

Background  

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) has been implicated in the attenuation of tumor metastasis by negatively regulating metalloproteinase (MMP) levels. RECK gene expression is downregulated in many solid tumors, with this downregulation being associated with poor prognosis. This study evaluated the role of RECK in cholangiocarcinoma (CCA).     

The bone disease associated with factor VIII deficiency in mice is secondary to increased bone resorption

Osteopenia and osteoporosis have increasingly become a recognized morbidity of factor VIII (FVIII) deficiency. Recently, we demonstrated that FVIII knockout (KO) mice had significantly decreased bone mass and bone strength despite the fact that they did not have haemarthroses. The aim of this study was to explore the mechanism of bone disease associated with FVIII deficiency. We compared biochemical markers of bone formation and osteoclastogenesis, inflammatory cytokines, as well as static and dynamic histomorphometry of genetically engineered FVIII KO male mice to those of wild‐type (WT) controls. At 20 weeks of age, FVIII KO mice, as well as WT controls, were sacrificed. Serum and bones were obtained at the time of sacrifice to study biochemical markers of bone formation (osteocalcin) and osteoclastogenesis (receptor activator of nuclear factor kappa‐β and osteoprotegerin), levels of inflammatory cytokines (interleukin‐1α and interferon‐β) and to perform static and dynamic histomorphometry of tibia cancellous bone. There was no difference in the biochemical markers of bone formation or osteoclastogenesis. However, there were differences in the two bone‐associated cytokines studied. In addition, histomorphometric examination revealed cancellous osteopenia in FVIII KO mice as evidenced by decreased bone area and trabecular number and increased trabecular separation. Bone formation parameters were normal in FVIII KO mice. In contrast, osteoclast‐lined bone perimeter was increased. These data demonstrate that bone disease in FVIII KO mice is due to an increased rate of bone resorption.     

Prematurity is associated with abnormal airway function in childhood

To evaluate the long-term effect of prematurity and/or hyaline membrane disease (HMD) on pulmonary function and airway reactivity, we studied 49 prematurely born children aged 10 to 13 years. They were divided into three groups according to birth weight and HMD status: Groups I and II comprised the children weighing less than 1,500 g at birth, and Group III those whose birth weight exceeded 1,500 g. Children without HMD at birth were classified as Group I and those with HMD as Group II or III. We performed both pulmonary function tests and methacholine (MCh) challenges and compared the results with those of 27 age-matched controls born at term. We found that FEV1 and RV/TLC ratios were significantly different from control values in the groups with birth weights less than 1,500 g, regardless of their HMD status (Groups I and II). In Group I, results for FEF25-75%, Vmax50%, and DLCO were lower than those of controls. Airway reactivity was significantly increased in Groups I and II. A 20% drop in FEV1 after MCh challenge was found in 88%, 62%, 53%, and 36% of children in Groups I, II, and III and controls, respectively, and a 35% drop in SGaw occurred in 87%, 88%, 53%, and 59%. We conclude that prematurity and not HMD per se leads to long-term pulmonary abnormalities and to an increase in nonspecific airway reactivity.     

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice

AIM:To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum.METHODS:The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria.Liver samples were obtained from mice sacrif iced at 6,8,10,14,and 18 wk after in-fection.Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining.The expression of osteopontin was determined with im-munohis...     

Insulin resistance is associated with a metabolic profile of altered protein metabolism in Chinese and Asian-Indian men

Aims/hypothesis

Insulin resistance (IR) is associated with obesity, but can also develop in individuals with normal body weight. We employed comprehensive profiling methods to identify metabolic events associated with IR, while controlling for obesity.

Methods

We selected 263 non-obese (BMI approximately 24 kg/m²) Asian-Indian and Chinese men from a large cross-sectional study carried out in Singapore. Individuals taking medication for diabetes or hyperlipidaemia were excluded. Participants were separated into lower and upper tertiles of IR based on HOMA indices of ≤1.06 or ≥1.93, respectively. MS-based metabolic profiling of acylcarnitines, amino acids and organic acids was combined with hormonal and cytokine profiling in all participants.

Results

After controlling for BMI, commonly accepted risk factors for IR, including circulating fatty acids and inflammatory cytokines, did not discriminate the upper and lower quartiles of insulin sensitivity in either Asian-Indian or Chinese men. Instead, IR was correlated with increased levels of alanine, proline, valine, leucine/isoleucine, phenylalanine, tyrosine, glutamate/glutamine and ornithine, and a cluster of branched-chain and related amino acids identified by principal components analysis. These changes were not due to increased protein intake by individuals in the upper quartile of IR. Increased abdominal adiposity and leptin, and decreased adiponectin and IGF-binding protein 1 were also correlated with IR.

Conclusions/interpretation

These findings demonstrate that perturbations in amino acid homeostasis, but not inflammatory markers or NEFAs, are associated with IR in individuals of relatively low body mass.     

A haemorrhagic platelet disorder associated with altered stimulus-response coupling and abnormal membrane phospholipid composition

Summary. Haemorrhagic diatheses due to platelet function defects are a heterogenous and poorly understood group of conditions. We report the investigation of a female with a lifelong history of epistaxes, haemarthroses, menorrhagia and persistent iron-deficiency anaemia. Although platelet numbers and morphology were normal, platelet function was abnormal both in vivo and in vitro . Skin bleeding time was prolonged and aggregation thresholds in platelet-rich plasma to a variety of weak and strong agonists were increased. Platelet granule contents were normal and membrane glycoproteins GpIb and GpIIIa were present in normal amounts. Polyphosphoinositide metabolism and phosphatidic acid generation were diminished in thrombinstimulated platelets, as was phosphorylation of the 47 kD substrate for protein kinase C and the 20 kD protein myosin light chain kinase, indicating impaired generation of the intracellular second messengers diacylglycerol and inositol trisphosphate due to diminished stimulated phospholipase C activity. Although intracellular free calcium, calmodulin activity and basal cAMP concentrations were normal, washed platelets showed increased cAMP accumulation following stimulation with prostaglandin E1 and forskolin. Platelet membrane lipid analysis revealed a reduction in plasmalogen phosphatidylethanolamine content. It is suggested that the membrane phospholipid abnormalities cause the abnormal platelet reactivity by interfering with signal transduction from platelet receptor, via intermediary G proteins, to phospholipase C and adenylate cylase. The bleeding tendency is likely to be a consequence of the altered stimulus-response coupling.     

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.

The cellular localization of the human papillomavirus (HPV)-16 E7 gene product in the cell lines CaSki and SiHa has been determined by both biochemical and immunocytochemical methods. These measurements show E7 to be localized in the cell nucleus, specifically with the nonchromatin nuclear structure or nuclear matrix. This localization of E7 required an unambiguous fractionation of the nuclear constituents. This was achieved by using a gentle sequential fractionation procedure to prepare the scaffold consisting of the nuclear matrix and intermediate filaments (NM-IF). Chromatin was cleaved with nuclease and the resulting nucleosomes eluted with 0.25 M ammonium sulfate. Immunostaining of cells after this extraction procedure with monoclonal antibodies (mAbs) to E7 revealed a fine grained, punctate nuclear fluorescence in CaSki and SiHa, which was absent in normal cervical keratinocytes and the HPV-negative cell line C33.1. Western blots of cell fractions with these mAbs showed that E7 was localized in the NM-IF fraction in SiHa and CaSki but was not detected in HPV-negative cells. A second protein of slightly higher mobility is identified by these antisera in HPV-16-containing cells. The data suggest that the previous inability to directly visualize E7 by immunocytology is due to the masking of epitopes by cellular components and not to low levels of protein.     

Low birthweight is associated with specific changes in muscle insulin-signalling protein expression

Aims/hypothesis People with low birthweight have an increased risk of developing type 2 diabetes mellitus in adulthood. The mechanistic basis of this phenomenon is not known. Here we investigate the effect of early growth restriction on the expression of insulin-signalling proteins in skeletal muscle in a human cohort and a rat model.Methods We recruited 20 young men with low birthweight (mean birthweight 2702±202 g) and 20 age-matched control subjects (mean birthweight 3801±99 g). Biopsies were obtained from the vastus lateralis muscle and protein expression of selected insulin-signalling proteins was determined. Rats used for this study were male offspring born to dams fed a standard (20%) protein diet or a low (8%) protein diet during pregnancy and lactation. Protein expression was determined in soleus muscle from adult offspring.Results Low-birthweight subjects showed reduced muscle expression of protein kinase C (PKC), p85, p110 and GLUT4. PKC, GLUT4 and p85 were also reduced in the muscle of rats fed a low-protein diet. Other proteins studied were unchanged in low-birthweight humans and in rats fed a low-protein diet when compared with control groups.Conclusions/interpretation We found decreased expression of specific insulin-signalling proteins in low-birthweight subjects compared to controls. These changes precede the onset of impaired glucose tolerance. The similarity of protein expression profile in the men with low birthweight compared to that of the offspring of rats fed a low-protein diet suggests that the rodent model is an accurate representation of the human situation. It also provides a potential mechanistic explanation as to why the fetal environment plays an important role in determining risk of developing type 2 diabetes.     

Osteomalacia and aplastic bone disease in aluminum-related osteodystrophy

The bone histology in patients with chronic renal failure and aluminum-related bone disease does not always show the excess accumulation of unmineralized osteoid (matrix) characteristic of osteomalacia. Frequently, bone aluminum accumulation is associated with normal or reduced amounts of unmineralized osteoid and low bone formation and is referred to as aplastic bone disease. In this study, we compared static and dynamic bone histomorphometric parameters and plasma PTH and aluminum levels in 12 patients with osteomalacia and 18 patients with aplastic bone disease who had been receiving dialysis for the same duration to determine if the difference in osteoid accumulation in these 2 lesions might be explained by differences in aluminum accumulation or PTH levels. The stainable bone surface aluminum level was significantly higher in the patients with osteomalacia compared to that in the group with aplastic bone [61 +/- 5% (+/- SEM) vs. 43 +/- 4%; P less than 0.02]. The rates of bone apposition and bone formation were lower in the group with osteomalacia (P less than 0.01). Plasma amino-terminal PTH was not significantly different in the 2 groups. The increment in plasma aluminum levels after a single infusion of deferoxamine was higher in the osteomalacic group than in the aplastic group, suggesting that the patients with osteomalacia accumulated more total body chelatable aluminum than did those with aplastic bone disease during a comparable length of time on dialysis. We conclude that the excess unmineralized osteoid in aluminum-related osteomalacia results from the high rate of total body aluminum accumulation, which directly causes uncoupling of matrix mineralization and matrix production, independent of PTH levels. Patients with aplastic bone disease who have accumulated lesser amounts of total body aluminum fail to develop excess unmineralized osteoid because production and mineralization of matrix are more closely coupled than in the osteomalacic lesion, despite a decline in osteoblast numbers.     

Increased formation of 8-iso-prostaglandin F(2alpha) is associated with altered bone metabolism and lower bone mass in hypercholesterolaemic subjects

OBJECTIVES: To investigate the relationship of 8-iso-prostaglandin (PG) F(2alpha) levels, a reliable marker of in vivo oxidative stress and lipid peroxidation, with bone mineral density (BMD), bone turnover markers, osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) in hypercholesterolaemia. DESIGN: Cross-sectional study. SETTING: University hospital centre. METHODS: Serum 8-iso-PGF(2alpha) levels were measured in 173 hypercholesterolaemic subjects and in 152 age- and sex-matched normocholesterolaemic controls. Femoral neck and lumbar spine BMD, serum bone-specific alkaline phosphatase (BAP), osteocalcin (OC), OPG and RANKL levels, as well as urinary levels of C-terminal telopeptides of type I collagen (CTX-I), were also assessed. RESULTS: Hypercholesterolaemic subjects showed higher (P < 0.0001) serum 8-iso-PGF(2alpha) levels than controls. They also had decreased (P < 0.0001) femoral neck and lumbar spine BMD, and lower (P < 0.0001) serum BAP and OC levels. No significant differences between hypercholesterolaemic and control subjects were found when comparing urinary CTX-I levels, or serum OPG and RANKL levels. In multivariate linear regression analysis, serum 8-iso-PGF(2alpha) was the only negative predictor for femoral neck BMD and serum BAP and OC levels in hypercholesterolaemic subjects. No significant correlation (all P > 0.25) was present between serum 8-iso-PGF(2alpha) levels and urinary CTX-I levels, or serum OPG and RANKL levels, in hypercholesterolaemic subjects. CONCLUSIONS: We found an association between increased serum 8-iso-PGF(2alpha) levels and lower bone mass and reduced serum BAP and OC concentrations in hypercholesterolaemic subjects. These results would suggest a possible role for oxidative stress in the development of lower bone mass in hypercholesterolaemia.     

Salt sensitivity in humans is associated with abnormal acid-base regulation

Metabolic acidosis has recently been observed in rat models of salt-sensitive genetic hypertension. To test the hypothesis that salt sensitivity in humans may be associated with abnormal acid-base homeostasis, we performed arterial blood gas analyses in young (20-31 years old) normotensive subjects (n = 40) who were placed on a low salt diet (20 mmol NaCl/day) for 2 weeks with either 200 mmol sodium chloride or placebo added to the low salt diet for 1 week each in a randomized, single-blind crossover order. Furthermore, a subset of the subjects (seven salt-sensitive and eight salt-resistant) received 200 mmol sodium/day as the citrate salt as a supplement to the low salt diet for a third week. During each regimen, blood pressure as well as arterial pH and bicarbonate levels were measured. Salt sensitivity was defined as a significant drop in mean arterial pressure greater than 3 mm Hg (mean of 30 readings taken during each diet, p less than 0.05) while the subject was on the low salt diet. According to this definition, 16 subjects were salt-sensitive and 24 salt-resistant. During the high sodium chloride regimen, arterial pH and bicarbonate levels were significantly lower in the salt-sensitive than in the salt-resistant group (p less than 0.0001). The increase in blood pressure caused by sodium chloride correlated inversely to the arterial pH (r = -0.57, p = 0.0002) and bicarbonate levels (r = -0.52, p = 0.0007) during the high salt diet. Sodium chloride increased mean arterial blood pressure in the salt-sensitive subjects; sodium citrate did not. Sodium citrate led to an increase in pH and bicarbonate levels in both groups. Our finding that a sodium chloride-induced rise in blood pressure is associated with lower arterial plasma pH and bicarbonate levels points to an abnormality in renal acid-base regulation in salt-sensitive subjects.     

Chronic ethanol-induced insulin resistance is associated with macrophage infiltration into adipose tissue and altered expression of adipocytokines

BACKGROUND: Chronic ethanol consumption disrupts glucose homeostasis and is associated with the development of insulin resistance. While adipose tissue and skeletal muscle are the two major organs utilizing glucose in response to insulin, the relative contribution of these two tissues to impaired glucose homeostasis during chronic ethanol feeding is not known. As other models of insulin resistance, such as obesity, are characterized by an infiltration of macrophages into adipose tissue, as well as changes in the expression of adipocytokines that play a central role in the regulation of insulin sensitivity, we hypothesized that chronic ethanol-induced insulin resistance would be associated with increased macrophage infiltration into adipose tissue and changes in the expression of adipocytokines by adipose tissue. METHODS: Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair-fed a control diet for 4 weeks. The effects of chronic ethanol feeding on insulin-stimulated glucose utilization were studied using the hyperinsulinemic-euglycemic clamp technique, coupled with the use of isotopic tracers. Further, macrophage infiltration into adipose tissue and expression of adipocytokines were also assessed after chronic ethanol feeding. RESULTS: Hyperinsulinemic-euglycemic clamp studies revealed that chronic ethanol feeding to rats decreased whole-body glucose utilization and decreased insulin-mediated suppression of hepatic glucose production. Chronic ethanol feeding decreased glucose uptake in epididymal, subcutaneous, and omental adipose tissue during the hyperinsulinemic-euglycemic clamp, but had no effect on glucose disposal in skeletal muscle. Chronic ethanol feeding increased the infiltration of macrophages into epididymal adipose tissue and changed the expression of mRNA for adipocytokines: expression of mRNA for monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin-6 were increased, while expression of mRNA for retinol binding protein 4 and adiponectin were decreased in epididymal adipose tissue. CONCLUSIONS: These data demonstrate that chronic ethanol feeding results in the development of insulin resistance, associated with impaired insulin-mediated suppression of hepatic glucose production and decreased insulin-stimulated glucose uptake into adipose tissue. Chronic ethanol-induced insulin resistance was associated with increased macrophage infiltration into adipose tissue, as well as changes in the expression of adipocytokines by adipose tissue.