Tumor necrosis factor-α increases alkaline phosphatase expression in vascular smooth muscle cells via MSX2 induction

Hye-Lim Leea, Kyung Mi Wooa, Hyun-Mo Ryooa and Jeong-Hwa Baek, a,

a Department of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea



Abstract
Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-α (TNF-α) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-α-mediated vascular calcification has not yet been fully defined. Therefore, in this study, we aimed to investigate whether MSX2 acts as a crucial regulator in TNF-α-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs).

TNF-α increased the expression of osteogenic marker genes including RUNX2, osterix, alkaline phosphatase (ALP), and bone sialoprotein, and it also promoted matrix mineralization in VSMCs. In addition, TNF-α enhanced MSX2 expression in a dose- and time-dependent manner. MSX2 over-expression alone induced ALP expression, whereas knockdown of MSX2 with small interfering RNA completely blocked TNF-α-induced ALP expression.

New protein synthesis was dispensable for MSX2 induction by TNF-α, and the inhibition of NF-κB by BAY-11-7082 or by dominant negative IκBα abolished the TNF-α-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression.

In conclusion, our study suggests that TNF-α directly induces MSX2 expression through the NF-κB pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs

Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients

Objective: Alkaline phosphatase (AP) attenuates inflammatory responses by lipopolysaccharide detoxification and may prevent organ damage during sepsis. To investigate the effect of AP in patients with severe sepsis or septic shock on acute kidney injury.

Design and Setting: A multicenter double-blind, randomized, placebo-controlled phase IIa study (2:1 ratio).

Patients: Thirty-six intensive care unit patients (20 men/16 women, mean age 58 ± 3 years) with a proven or suspected Gram-negative bacterial infection, ≥2 systemic inflammatory response syndrome criteria (<24 hours), and <12 hours end-organ dysfunction onset were included.

Intervention: An initial bolus intravenous injection (67.5 U/kg body weight) over 10 minutes of AP or placebo, followed by continuous infusion (132.5 U/kg) over the following 23 hours and 50 minutes.

Measurements and Main Results: Median plasma creatinine levels declined significantly from 91 (73-138) to 70 (60-92) μmol/L only after AP treatment. Pathophysiology of nitric oxide (NO) production and subsequent renal damage were assessed in a subgroup of 15 patients. A 42-fold induction (vs. healthy subjects) in renal inducible NO synthase expression was reduced by 80% ± 5% after AP treatment. In AP-treated patients, the increase in cumulative urinary NO metabolite excretion was attenuated, whereas the opposite occurred after placebo. Reduced excretion of NO metabolites correlated with the proximal tubule injury marker glutathione S-transferase A1-1 in urine, which decreased by 70 (50-80)% in AP-treated patients compared with an increase by 200 (45-525)% in placebo-treated patients.

Conclusions: In severe sepsis and septic shock, infusion of AP inhibits the upregulation of renal inducible NO synthase, leading to subsequent reduced NO metabolite production, and attenuated tubular enzymuria. This mechanism may account for the observed improvement in renal function.


Critical Care Medicine:
February 2009 - Volume 37 - Issue 2 - pp 417-e1Heemskerk, Suzanne PhD; Masereeuw, Rosalinde PhD; Moesker, Olof; Bouw, Martijn P. W. J. M.; van der Hoeven, Johannes G. MD, PhD; Peters, Wilbert H. M. PhD; Russel, Frans G. M. PhD; Pickkers, Peter MD, PhD; on behalf of the APSEP Study Group

Effect of alendronate on bone mineral density and bone turnover markers in post-gastrectomy osteoporotic patients

Alendronate decreases the urinary levels of cross-linked N-terminal telopeptides of type I collagen (NTX; about 45% at 3 months) and serum levels of alkaline phosphatase (ALP; about 27% at 24 months), leading to an increase in lumbar spine bone mineral density (BMD; about 9% at 24 months) in postmenopausal Japanese women with osteoporosis. However, the effectiveness of oral bisphosphonates on osteoporosis remains to be established in patients who have undergone a gastrectomy. The objective of the present case series study was to examine the effect of alendronate on BMD and bone turnover markers in post-gastrectomy osteoporotic patients.

Sixteen patients (3 men and 13 postmenopausal women) with osteoporosis, who had undergone a gastrectomy (mean age: 69.1 years), were recruited in our outpatient clinic. All the patients were treated with alendronate (5 mg daily or 35 mg weekly) for 24 months. The effects of alendronate on lumbar spine (women) or total hip (men) BMD and urinary NTX and serum ALP levels were examined. A total or partial gastrectomy had been performed for eight patients each. The mean duration after surgery was 16.0 years. With alendronate therapy, urinary NTX levels significantly decreased at 3 months (-27.0%). Serum ALP levels decreased (-12.1%) and lumbar spine BMD increased (+5.2%), but total hip BMD did not significantly change (+0.6%) at 24 months. No severe adverse events were observed, and alendronate therapy was well tolerated. These results suggest that alendronate mildly increases lumbar spine BMD by mildly reducing bone turnover in osteoporotic patients after a gastrectomy.

Iwamoto J, Uzawa M, Sato Y, Takeda T, Matsumoto H.
Institute for Integrated Sports Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

Intestinal alkaline phosphatase regulates protective surface microclimate pH

Keio U School of Medicine;

Regulation of localized extracellular pH (pHe) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pHe due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR-dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y1 receptor to the brush border membrane of duodenal villi. The P2Y1 agonist 2-methylthio-ADP increased DBS, whereas the P2Y1 antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor L-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y1 antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pHe, increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP/P2Y/HCO3- surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Microfluidic immunosensor design for the quantification of interleukin-6 in human serum samples

Interleukin-6 (IL-6), an inflammatory cytokine, is one of the most important mediators of fever, the acute phase response, and inflammatory conditions. Described here is an integrated microfluidic immunosensor capable of detecting the concentration of IL-6 in human serum samples by use of an electrochemical method in a microfluidic biochip format. The detection of IL-6 was carried out using a sandwich immunoassay method based on the use of anti-IL-6 monoclonal antibodies, immobilized on a 3-aminopropyl-modified controlled-pore glass (APCPG) packet in a central channel (CC) of the microfluidic system. The IL-6 in the serum sample is allowed to react immunologically with the immobilized anti-IL-6 and biotin-labeled second antibodies specific to IL-6. After washing, the streptavidin–alkaline phosphatase conjugate is added. p-Aminophenyl phosphate is converted to p-aminophenol by alkaline phosphatase, and the electroactive product is quantified on a gold electrode at 0.10 V. For electrochemical detection and enzyme immunoassay, the LOD was 0.41 and 1.56 pg mL−1, respectively. Reproducibility assays employed repetitive standards of IL-6, and the intra- and inter-assay coefficients of variation were below 6.5%. Compared with the traditional IL-6 sensing method, the integrated microfluidic immunosensor required smaller amounts of sample to perform faster detection.

ARTICLE

Staphylococcal enterotoxin C injection in combination with ascorbic acid promotes the differentiation of bone marrow-derived mesenchymal stem cells...

Staphylococcal enterotoxin C injection is established as a clinical therapy for delayed healing or disunion of bone fractures. In the present study, the effects of staphylococcal enterotoxin C injection in combination with ascorbic acid (SEC-AA) on the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) and their influences on the mineralization of osteoblasts were investigated. SEC-AA treatment induced increased levels of alkaline phosphatase activity in MSCs and increased numbers of alizarin red-stained calcified nodules, indicating enhanced differentiation of MSCs into osteoblasts. The findings demonstrated that SEC-AA promoted the differentiation of MSCs into osteoblasts and accelerated the cytopoiesis of osteoblasts. Our data provide a cytological model for bone fracture therapy aimed at shortening the time required for healing and improving the clinical outcome, and also provide a theoretical basis for inducible differentiation of MSCs, mineralization of osteoblasts and reconstruction of bone tissues.

ARTICLE

Liver function tests

Exam Overview
Some blood tests are used to determine whether your liver is damaged or inflamed. Although these tests help your doctor evaluate how well your liver is working, they cannot tell if you have hepatitis C.

Tests that assess liver function
Your doctor may do tests to measure certain chemicals produced by the liver. These tests can help your doctor check how well your liver is working. Tests may measure:

Bilirubin

Albumin

Prothrombin time (a measure of blood clotting). It may also be called International Normalized Ratio (INR).

Tests that check for inflammation of the liver (liver enzyme studies)
If you have increased levels of the following, your liver may be damaged:

Alanine aminotransferase (ALT or SGPT)

Aspartate aminotransferase (AST or SGOT)

An increased level of alkaline phosphatase (AP) may indicate blockage of bile ducts.

Why It Is Done
Liver tests are done when a medical history or physical exam suggests that something may be wrong with your liver.

These tests can also help diagnose long-term (chronic) infection. Hepatitis C infection is considered chronic when liver enzymes remain elevated for longer than 6 months.

If you are being treated with antiviral therapy, you may have liver tests from time to time to see whether treatment is working.

Results
Findings of liver function tests may include the following:

Normal
All levels are within the normal range.

Abnormal
One or more levels are outside the normal range. Abnormal liver function tests may indicate that your liver is inflamed or is not working normally. This can be a sign that you have a viral infection.

What To Think About
Elevated liver enzymes can be caused by many things other than hepatitis C, such as obesity, hepatitis B, autoimmune hepatitis, certain medicines, or long-term alcohol use. So you will need other tests (such as a hepatitis C antibody blood test or a liver biopsy) to confirm a diagnosis of hepatitis C.

People with chronic hepatitis C have abnormal liver enzyme levels most of the time. But the levels can fluctuate between normal and abnormal throughout the course of the disease.

Liver tests can be used to help you and your doctor develop a treatment plan. Signs that you might need treatment include:

Liver enzyme levels that remain above normal for longer than 6 months, which is evidence of chronic infection.
Detectable levels of hepatitis C virus in your blood (positive hepatitis C RNA test). This is a sign of an active infection.
Evidence of serious liver damage. This is detected with a liver biopsy.