Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (ß-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by ß-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification.

Protective effects of Vitamin C against spinal cord injury-induced renal damage through suppression of NF-κB and proinflammatory cytokines.

Spinal cord injury [SCI] leads to complex cellular and molecular interactions which affects various organ systems. The present study focused on determining the protection offered by Vitamin C against spinal injury-induced kidney damage in wistar rats. The experimental protocol was performed with three groups; Sham, SCI and Vitamin C [20 mg/kg/bw] followed by SCI. The kidney tissue was investigated for oxidative stress parameters [reactive oxygen species, protein carbonyl, sulphydryl content, thiobarbituric acid reactive species [TBARS], and myeloperoxidase activity] and antioxidant status [glutathione, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase activity]. Further, inflammation studies were performed by analyzing expression of NF-κB, cycloxygenase-2, iNOS through western blot analysis and inflammatory cytokines by TNF-α and IL-1ß levels. The present study shows clear evidence that Vitamin C treatment abrogated spinal injury-induced oxidative stress and inflammatory responses and enhanced the antioxidant status. Thus, the protection offered by Vitamin C against spinal cord injury-induced kidney damage is attributed to its anti-oxidant and anti-inflammatory effects.

[In vitro effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on differentiation from human adipose-derived mesenchymal stem cells to endothelial cells].

OBJECTIVE: To explore the effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide(W7) on the differentiation from human adipose-derived mesenchymal stem cells (hADSCs) to endothelial cells. METHODS: hADSCs were cultured with serum-free differential medium containing 40 ng/ml vascular endothelial growth factor (VEGF) and 10ng/ml basic fibroblast growth factor (bFGF). Cells were divided into control group (differential medium without W7), high-dose group (containing 30 µmol/L W7), medium-dose group (containing 20 µmol/L W7), and low-dose group ( containing 10 µmol/L W7). The hADSCs were cultured for 8 days, and then the changes in the phenotypes of von Willebrand factor (vWF) and vessel-selective cadherin (VE-Cadherin) were detected by flow cytometry (FCM). The intracellular Ca(2+) labeled with Fluo-3 was detected by laser confocal microscopy. After hADSCs planting on Matrigel, their angiogenic potentials were observed under the inverted phase contrast microscope, and the expression of extracellular regulated kinase (ERK) and phosphorylated extracellular regulated kinase (p-ERK) were evaluated by Western blot. RESULTS: After the hADSCs were cultured for 8 days, compared with the control group, the expressions of vWF and VE-Cadherin significantly increased along with the decrease of W7 level and the intracellular Ca(2+) also significantly increased (Pü0.01). Lumina-like vascular structure was formed in W7 treatment groups, but not in the blank control group. Compared with the blank control group, the expression of ERK showed no significant in W7 treatment groups (high-, medium-, and low-dose groups)(P>0.05); however, along with the decrease of W7 levels, the expression of p-ERK significantly increased(P<0.05). CONCLUSION: W7 in proper levels can effectively induce the differentiation from hADSCs to endothelium by increasing the intracellular Ca(2+) level and thus activating the ERK/MAPK pathway.

The circulating level of miR-122 is a potential risk factor for endothelial dysfunction in young patients with essential hypertension.

MicroRNAs are key molecules involved in the regulation of endothelial function. They are important risk factors and biomarkers for the development of hypertension related to endothelial dysfunction. However, the gene expression patterns associated with hypertension development related to endothelial dysfunction have not been fully elucidated. We conducted a case-control study of 65 patients with essential hypertension (EH) and 61 controls without EH. Plasma levels of miR-122 and its target protein high-affinity cationic amino acid transporter 1 (CAT-1) were measured by qRT-PCR and ELISA, respectively. miR-122 expression in plasma of patients with EH was significantly higher than that of the control group (p = 0.001), while CAT-1 expression in patients with EH was significantly lower than that in the control group (p = 0.018). miR-122 expression in plasma of young patients with EH was significantly higher than that in young people without EH (p = 0.0004), and CAT-1 expression in plasma of young patients with EH was also significantly lower than that of the control group (p = 0.002). CAT-1 expression in the plasma of young participants was significantly higher than that of individuals aged ≥40 years (p = 0.003), whereas miR-122 expression was significantly lower (p = 0.001). We showed that among patients with EH, the high expression of miR-122 contributed to endothelial dysfunction by suppressing the expression of the CAT-1 protein, which led to a decrease in CAT-1 expression in plasma. Therefore, high expression of miR-122 appears to be a risk factor for endothelial dysfunction in EH, especially in younger patients.

Soluble angiopoietin receptor Tie-2 in patients with acute myocardial infarction and its detection by optical protein-chip.

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P<0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

[Influence of breast carcinoma cells on normal endothelial cells: experimental study with a co-culture system].

OBJECTIVE: To study the influence of breast carcinoma cells on normal endothelial cells. METHODS: Human endothelial cells were isolated from umbilical cord blood. Medium Z-MCF-7-EC was established and was used to co-culture the normal endothelium cells (ECs) and human breast cancer cells of the line MCF-7. Normal endothelial cells cultured by itself were used as control. Light microscopy and transmission electron microscopy were used to observe the morphology of the 2 kinds of endothelial cells. Expression of the genes ESM, IGFBP-3, alphavbeta3, VE-C, and Tie-2-2 was analyzed by semi-quantitative RT-PCR using a house-keeping gene beta(2m) as inner reference gene. RESULTS: The ECs co-cultured with MCF-7 breast cancer cells were abnormal in shape with increased size of nucleus and nucleolus, increased size ratio of nuclear to nucleoplasm, increased depth of surface fenestration, loosed and distorted endoplasm, increased size of cell-cell junctions, decreased number of surface microvilli, and tubules formed by ECs. The expression values of the genes ESM, IGFBP-3, alphavbeta3, VE-C, and Tie-2-2 in the ECs co-cultured with MCF-7 breast cancer cells were all up-regulated in comparison with those in the controls (P < 0.01, P < 0.05, P < 0.01, P < 0.01, and P < 0.05 respectively). CONCLUSION: Breast cancer cells promote formation of new vessels with endothelial cells different from the normal ECs in character and behavior.

[Serum inflammatory related cytokines and circulating endothelial cells in patients with acute coronary syndrome].

OBJECTIVE: To study the number of CECs in patients with acute myocardial infarction (AMI) and unstable angina (UA), and to investigate its relationship with inflammatory related cytokines. METHODS: 37 patients with AMI, 12 patients with UA, and 42 health controls were studied. CECs were isolated from peripheral blood by using of immunomagnetic beads coated with antibodies against CD146. Their endothelial origin was confirmed by the positive labelling of von Willebrand Factor (vWF), CD31 and electron microscope. Annexin V-FITC/PI kit was used to measure the apoptosis of CECs. Inflammatory related cytokines were analyzed turbidimetrically or ELISA using of commercially available testing kit. RESULTS: CECs number was significantly higher in AMI and UA patients [medians (interquartile range) were 52 (28 approximately 81.5) cells/ml and 29 (18 approximately 61) cells/ml respectively] compared with health control [10.5 (6-16.5)cells/ml, P < 0.001]. After excluding diabetes patients, the number of CECs and CRP in AMI and UA group (n = 26) were still significantly higher than controls. The necrotic rate of CECs in AMI and UA was significantly higher than controls (P < 0.01). Correlation analysis revealed a significant positive correlation between CECs and CRP, or IL-6 (r = 0.677, 0.316, P = 0.000, 0.002). The multivariate linear regression analysis showed that CRP and Diabetes increased the number of CECs significantly (OR = 0.620, 0.164, 95% CI 3.985-6.751, 0.301-21.877, P = 0.000, 0.044). CONCLUSION: The mechanism responsible for the increase of CECs in acute coronary disease may be due to the vessel injury caused by inflammation.

Micro-vascular medicine and proteomics.

A new technique is sweeping the world, and changing the course of human work and life. It is impacting upon models, methods and the development of medical research. In the development of this new technique, a huge quantity of experimental research and clinical practice has proved that many human diseases have a close relationship to pathological changes that take place in the microvascular system. It has been proven that the microvascular system is the target for studying disease development and the treatment of disease. Many studies have shown that successful pathogenesis and pathological research must be aimed at understanding the key proteins in cells, organs and systems, as well as investigating their interaction, and finding out how these proteins change under disease conditions. This paper reviews the current status of microvascular medicine and proteomics.

The effects of nimodipine on regional cerebral blood flow, brain water and electrolyte contents in rats with subarachnoid hemorrhage.

Secondary cerebral ischemic injury is a major cause of mortality and disability from subarachnoid hemorrhage (SAH). In this study, the protective effects of nimodipine were investigated. Rat SAH models were divided into a sham-operated group, a saline-controlled, and a nimodipine-treated group by an endovascular piercing method. Nimodipine, 100 microg/kg BW was injected intraperitoneally 30 minutes before operation and was repeated every 6 hours. Dynamic changes in cortical regional cerebral blood flow (rCBF) using a laser Doppler flow-meter probe, and somatosensory evoked potentials (SEP) were estimated. Brain water content, sodium, potassium and calcium contents at different time points were determined. rCBF, latency of SEP, brain water and electrolyte contents did not statistically change in sham-operated rats. In saline-controlled rats, rCBF decreased immediately after SAH, and stabilized at low levels within 24 hours. The latency of SEP delayed gradually after SAH. Brain water and sodium increased, while potassium decreased at 6 hours and 24 hours. Brain calcium content increased significantly from 1 hour to 24 hours after induction of SAH. Extents of alterations of the above parameters caused by SAH in the nimodipine-treated group were less than those in the saline-controlled group, statistically. In conclusion, nimodipine partly prevents a decrease in cerebral blood supply and attenuates secondary cerebral ischemic injury after SAH.

L-arginine improves cerebral blood perfusion and vasomotion of microvessels following subarachnoid hemorrhage in rats.

The purpose of this study is to investigate the effect of L-arginine (L-Arg) on cerebral blood perfusion and vasomotion (perfusion motion) in microvessels following subarachnoid hemorrhage (SAH). Rat noncraniotomy SAH models were used and animals were divided into sham-operated, saline-treated, and L-Arg-treated groups. L-Arg was injected intraperitoneally 30 minutes before the operation and repeated every 6 hours, with a single dose of 0.5 g/kg bw. Dynamic changes in regional cerebral blood flow (CBF) and vasomotion within 24 hours were measured using a laser Doppler flow-meter probe. Serum nitric oxide (nitrite/nitrate) and plasma endothelin-1 levels were also measured at different time points within 24 hours. Morphologic changes in neurons in the hippocampus CA1 region were examined. SAH gave rise to an immediate and persistent decrease in CBF in saline-treated rats. Abnormal vasomotions with decreased frequency and amplitude were observed. Serum nitric oxide decreased, while plasma endothelin-1 increased significantly. Neurons in the hippocampus CA1 region were severely damaged. The above pathological alterations in the L-Arg-treated group were alleviated. It was concluded that L-Arg, which increases cerebral blood perfusion and improves vasomotions of microvessels by enhancing nitric oxide levels and decreasing endothelin-1 levels in blood, exerts a protective effect on secondary cerebral ischemic injury following experimental SAH.

Small-size circulating endothelial microparticles in coronary artery disease.

OBJECTIVES: Several recent lines of evidence indicate that endothelial microparticles are a new biomarker that can be used to monitor endothelial dysfunction in coronary artery disease (CAD). However, data concerning the detection of small microparticles (diameter <0.5 µm) are lacking. The aim of this study was to detect small-size endothelial microparticles (SEMPs) in CAD patients to monitor endothelial dysfunction. METHODS: In total, 19 CAD patients and 14 healthy subjects were recruited. The absolute numbers and percentages of CD31(+)/CD42b- SEMPs and CD62E(+) SEMPs were determined by flow cytometry. Clinical parameters were also recorded. RESULTS: The mean percentage of CD62E(+) SEMPs was higher in the CAD patient group than in the healthy subject group. The area under the receiver operating characteristic curve of the percentage of CD62E(+) SEMPs was 0.795, and the cut-off value was 1.35. There was no correlation between the percentage of CD62E(+) SEMPs and various clinical parameters. CONCLUSION: The percentage of CD62E(+) SEMPs is a potential biomarker for monitoring endothelial function in CAD.

Circulating CD62P small microparticles levels are increased in hypertension.

The study aims to find a new biomarker in hypertension. Our study is the first time to demonstrate that the CD62P small microparticle (diameter is <0.5 um) was a new microparticle population and a new biomarker in hypertension.

Automated method for tracking vasomotion of intravital microvascular and microlymphatic vessels.

Dynamic tracking of microvascular and microlymphatic vasomotion presents a critical image processing technique in the evaluation of function and dysfunction of the microvasculature. Many methods for determination of diameter changes have been reported. Previous methods which were specifically developed for vasomotion tracking of intravital, fluorescence-free, rapidly constricting microvascular and microlymphatic vessels have various limitations due to complex image background, vessel wall distortion, image drift, noise and other artifacts. In order to overcome these major obstacles and remove undesirable limitations, this study proposed a tracking strategy based on feature matching of moving object-VasTrack. First, we calculate the image drift vector by feature template matching of a landmark in the background. Second, dynamically regulate the position of a sample line in accordance with the drift vector for sustaining the consistency of measurement location. Third, dynamically recognize and track edge position changes by applying feature template matching using edge context. This method does not require special preprocessing of video image registration and rotation. VasTrack compensates efficiently for image drift and vessel wall distortion, can simultaneously track and determine vessel diameters at any orientation and in multiple locations. Testing proved that VasTrack is robust and accurate, and will satisfy the needs of basic microcirculatory research and clinical inspection.