The comparison of methods to identify the presence of fibrocytes in formalin-fixed, paraffin-embedded archival cardiac tissue with coronary heart disease.

The purpose of this study was to find the optimal technical approach to identify the presence of fibrocytes in formalin-fixed, paraffin-embedded archival cardiac tissue with CHD (coronary heart disease). Using the coexpression markers CD45 and αSMA, the presence of fibrocytes was examined by three different methods, including double immunohistochemistry staining, combination labeling of immunohistochemistry and immunofluorescence and double immunofluorescence labeling. Double immunohistochemistry staining was very difficult to identify the CD45(+)/αSMA(+) fibrocytes. Although combination staining of immunohistochemistry and immunofluorescence has made it possible to evaluate the co-localization of CD45 and αSMA in the fibrocytes, this method was prone to produce many false positive cells. In contrast, CD45(+)/αSMA(+) fibrocytes could be clearly recognized by double immunofluorescence labeling. In conclusion, double immunofluorescence labeling is the optimal technical approach to identify the presence of fibrocytes in routinely processed cardiac tissue with CHD.

Extracorporeal cardiac shock wave therapy ameliorates myocardial fibrosis by decreasing the amount of fibrocytes after acute myocardial infarction in pigs.

OBJECTIVES: Extracorporeal shock wave (SW) therapy ameliorates cardiac remodeling after acute myocardial infarction (AMI). However, it remains to be examined whether and how SW therapy ameliorates myocardial fibrosis after AMI. Fibrocytes are associated with myocardial fibrosis. Thus, we examined whether SW therapy ameliorates myocardial fibrosis and whether fibrocytes are associated after AMI in pigs. MATERIALS AND METHODS: AMI was created by coronary embolism. Twenty-five pigs were divided into three groups: AMI+SW group (AMI with SW therapy, n=15), AMI group (without SW therapy, n=5), and sham+SW group (SW therapy without AMI, n=5). The collagen area fraction was examined by Masson's trichrome staining. The presence of fibrocytes was identified by immunofluorescence and confocal microscopy. The location of CXCL12 was examined by immunohistochemistry. RESULTS: Compared with the AMI group, the AMI+SW group showed significantly ameliorated myocardial fibrosis in terms of collagen area fraction (27.21±8.13 vs. 10.13±4.96, P<0.05) and reduced fibrocytes (CD34/α-smooth muscle actin: 35.40±11.72 vs. 12.27±7.71, P<0.05; CXCR4/α-smooth muscle actin: 40.80±8.96 vs. 16.54±6.38, P<0.05). There were positive correlations between the collagen area fraction and the number of fibrocytes (r=0.936; P<0.05) and between the number of CXCR4 fibrocytes and the SDF-1/CXCL12 cells (r=0.802; P<0.05) in the three groups. CONCLUSION: The results show that SW therapy ameliorates myocardial fibrosis after AMI in pigs, which is associated with the decreased amount of fibrocytes.

Fibrocytes are associated with the fibrosis of coronary heart disease.

Fibrocytes contribute significantly to fibrosis in many cardiac diseases. However, it is not clear whether fibrocytes are associated with the fibrosis in coronary heart disease (CHD). The aim of this study was to determine whether fibrocytes are involved in cardiac fibrosis in CHD. We identified the presence of fibrocytes in CHD heart by immunofluorescence and confocal microscopy, examined the collagen volume fraction by Masson's Trichrome staining, and evaluated the correlation between fibrocytes and cardiac fibrosis. In conjunction, we examined the location of CXCL12, a homing factor and specific ligand for CXCR4, by immunohistochemistry. Fibrocytes were identified in 26 out of 27 CHD hearts and in 10 out of 11 normal hearts. Combinations, including CD34/αSMA, CD34/procollagen-I, CD45/αSMA, CXCR4/procollagen-I and CXCR4/αSMA, stained significantly more fibrocytes in CHD hearts as compared with those in normal hearts (p<0.05). There were positive correlations between the collagen volume fraction and the amount of fibrocytes (r=0.558; p=0.003<0.01) and between the number of CXCR4(+) fibrocytes and the CXCL12(+) cells (r=0.741; p=0.000<0.01) in CHD hearts. Based upon these findings, we conclude that fibrocytes, likely recruited through the CXCR4/CXCL12 axis, may contribute to the increase in the fibroblast population in CHD heart.

Therapeutic effect of intravenous bone marrow-derived mesenchymal stem cell transplantation on early-stage LPS-induced acute lung injury in mice.

OBJECTIVE: To investigate the effect of intravenous bone marrow-derived mesenchymal stem cell (MSC) transplantation for early intervention of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. METHODS: Thirty-six mice were randomized into control group, PBS-treated ALI group, and MSC-treated ALI group. In the latter two groups, mouse models of ALI were established by intranasal instillation of LPS, and 1 h later, the 4th passage of MSCs isolated from the bone marrow of mice or PBS were administered via the tail vein. The histological findings, lung wet/dry (W/D) weight ratio, neutrophil count and protein and cytokine contents in the bronchoalveolar lavage fluid (BALF), and myeloperoxidase (MPO) level in the lung tissue were analyzed at 24 h after MSC administration. Engraftment of MSCs in the recipient lung was determined by fluorescent PKH26 staining and flow cytometry. RESULTS: Compared with the control group, PBS-treated ALI group showed significantly higher protein levels, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and neutrophil count in the BALF and MPO content in the lung tissue, with also severe damage of lung histology. MSCs administration significantly reduced the lung W/D weight ratio, the levels of protein, TNF-α, IL-6 and neutrophil count in the BALF and MPO content in the lung tissue, and obviously lessened the lung injury 24 h after the transplantation. MSC administration also significantly increased the level of IL-10 in the BALF. CONCLUSION: Intravenous MSC transplantation can effectively improve the lung histology, attenuate the inflammatory response, reduce pulmonary edema in the early stage of LPS-induced ALI in mice, and such effects are independent of MSC engraftment in the lungs.

Role of asymmetric dimethylarginine in acute lung injury induced by cerebral ischemia/reperfusion injury in rats.

OBJECTIVE: To determine the role of asymmetric dimethylarginine (ADMA) in acute lung injury induced by cerebral ischemia/reperfusion (I/R) injury in rats. METHODS: Adult male SD rats were randomly divided into 4 groups, namely the sham-operated group (S), cerebral I/R model group, ADMA+I/R group, and dimethylarginine dimethylaminohydrolase (DDAH)+I/R group. In the latter 3 groups, acute lung injury was induced by left middle cerebral artery occlusion for 120 min. After a 24-h reperfusion, the rats were sacrificed and the activities of nitric oxide synthase (NOS) and contents of nitric oxide (NO) were measured using reductase and colorimetric assay. The mRNA and protein expressions of protein kinase C (PKC) and myosin light chain kinase (MLCK) in the lung tissues were detected with RT-PCR and Western blotting, respectively. The contents of ADMA in the bronchoalveolar lavage fluid (BALF) and blood flowing into and out of the lungs were measured by ELISA. RESULTS: Cerebral I/R injury caused significantly elevated ADMA levels in the BALF and blood flowing into the lungs, and obviously lowered the NO concentration and NOS activity in the lung tissues (P<0.05). Following cerebral I/R injury, MLCK and PKC mRNA and protein expressions were significantly upregualted in the lung tissues (P<0.05). Exogenous DDAH obviously decreased the levels of ADMA in the BALF and blood flowing into the lungs, increased NO concentration and NOS activity, and down-regulated MLCK and PKC mRNA and protein expressions in lung tissues of rats with cerebral I/R injury (P<0.05). CONCLUSION: ADMA contributes to the development of acute lung injury following cerebral I/R injury in rats by upregulating MLCK and PKC expression. ADMA may serve as a novel therapeutic biomarker and a potential therapeutic target for acute lung injury induced by cerebral I/R injury.

Effect of asymmetric dimethylarginine on MIF expression and TNF-α and IL-8 secretion in THP-1 monocytes-derived macrophages.

OBJECTIVE: To investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-α (TNF-α) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-α and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments. RESULTS: ADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-α and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA. CONCLUSION: ADMA can up-regulate MIF expression and induce TNF-α and IL-8 secretion in THP-1 monocyte-derived macrophages.

Asymmetric dimethylarginine upregulates the expression of ACAT-1 in THP-1 macrophage-derived foam cells.

OBJECTIVE: To investigate the effects of asymmetric dimethylarginine (ADMA) on ACAT-1 expression and cholesterol content in THP-1-derived macrophages and foam cells. METHODS: THP-1 cells were induced to differentiate into macrophages and further into foam cells. The macrophages and foam cells were exposed to different concentrations (0, 3.75, 7.5, 15, and 30 µmol/L) of ADMA for varying time lengths (6, 12, and 24 h), and the changes in ACAT-1 mRNA and protein levels in the cells were measured with RT-PCR and Western blotting. The cellular cholesterol content was measured with enzyme-linked colorimetry assay. RESULTS: In THP-1-derived macrophages and foam cells, the expression levels of ACAT-1 mRNA and protein and cellular cholesterol content increased significantly in response to ADMA treatment in a time- and concentration-dependent manner. CONCLUSION: ADMA may play an important role in inducing foam cell formation from macrophages. ACAT-1 inhibition targeting the macrophages and foam cells may serve as a potential therapeutic target in the treatment of atherosclerosis.

[Change of cytochrome c in postconditioning attenuating ischemia-reperfusion-induced mucosal apoptosis in rat intestine].

The present study aimed to investigate the change of cytochrome c in postconditioning-attenuated ischemia-reperfusion (I/R)-induced mucosal apoptosis in rat intestine compared with ischemic preconditioning (IPC). Using rat model of intestine I/R injury, male Sprague-Dawley rats weighing 220-250 g were divided into 4 groups which were Sham operation group, I/R group, IPC group and ischemic postconditioning (IPOST) group. In these groups, I/R procedure was performed by the occlusion of the superior mesenteric artery (SMA) for 45 min followed by reperfusion for 1 h. In Sham group, there was no intervention. In IPC group, SMA was occluded for 5 min and reperfused for 5 min, for two cycles, before the prolonged occlusion. In IPOST group, three cycles of 30-s reperfusion and 30-s reocclusion were preceded at the start of reperfusion. After the reperfusion, the small intestines were sampled for experimental detection. Intestinal mucosal mitochondrial membrane potential was detected by confocal laser scanning microscopy. Expressions of cytochrome c and caspase-3 proteins were detected using Western-blot method. The apoptosis of intestinal mucosal cells was determined with agarose gel electrophoresis and deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) technique. Compared with I/R group, the mitochondrial membrane potentials and the expressions of cytochrome c protein were significantly increased, while the expressions of caspase-3 and the apoptotic rates were decreased in IPOST and IPC groups (P<0.05). There were no significant differences between IPOST and IPC groups (P>0.05). These data provide substantial evidence that IPOST attenuates I/R-induced mucosal apoptosis by reducing the release of cytochrome c from mitochondria in the rat small intestine.

[Role of nuclear factor-kappaB in apoptosis pathway of HUVEC].

AIM: To investigate the role of nuclear factor-kappaB in apoptosis pathway of HUVEC. METHODS: The cell lines of HUVEC cultured in vitro were divided into three groups: normal control group, Ang II group, and Gliotoxin group. We investigated the effects of Ang II (0.01 micromol/L, 0.1 micromol/L, 1 micromol/L and 10 micromol/L) on the viability of HUVEC with modified MTT. Then agarose gel electrophoresis and flow cytometry were applied to detect the apoptosis of HUVEC. Finally, the nuclear translocation of NF-kappaB subunit p65 was evaluated by immunocytochemistry. RESULTS: The viability of HUVEC decreased significantly after incubated with 10 micromol/L Ang II for 24 hours. The results of DNA agarose gel and flow cytometry showed that 10 micromol/L Ang II induced the apoptosis of HUVEC, and the apoptosis rate was significantlyhigher than normal control group (P < 0.05). 0.1 mg/L Gliotoxin antagonized this effect of Ang II. The results of immunocytochemistry suggested that NF-kappaB was activated in HUVEC induced by 10 micromol/L Ang II. In contrast, Gliotoxin inhibited the activation of NF-kappaB in HUVEC induced by Ang II. CONCLUSION: (1) Ang II can induce the apoptosis of HUVEC, while the inhibitor of NF-kappaB, Gliotoxin, can antagonize the effect of Ang II. (2) NF-kappaB may play an important role in apoptosis pathway of HUVEC induced by Ang II.

Copper-aspirin complex inhibits cyclooxygenase-2 more selectively than aspirin.

The antiinflammatory effects of the copper-aspirin complex (Cu-Asp) were more potent than that of Asp in rats or mice with fewer classic adverse effects. The aim of this study was to determine the cause by evaluating Cu-Asp selective inhibition on cyclooxygenases (COX). COX-1 inhibition was evaluated based on 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) in an endothelial cell model, and COX-2 inhibition was based on prostaglandin E(2) (PGE(2)) in a macrophage model. Radioimmunoassay (RIA) was applied to determine 6-keto-PGF(1alpha) in resting human umbilical vein endothelial cell line (ECV304), and PGE(2) in activated macrophages. The results showed that the inhibition of 6-keto-PGF(1alpha) yield by Cu-Asp (3 to 0.01 mM) was markedly weaker than that by aspirin (Asp); while the inhibition of PGE(2) yield by Cu-Asp (10 to 0.1 mM) was significantly stronger than that by Asp. Based on the inhibition on 6-keto-PGF(1alpha) and PGE(2), the medium inhibitory concentration (IC(50)) of Cu-Asp on COX-1 and on COX-2 was 1.03+/-0.15 mM, and 0.32+/-0.04 mM, respectively. The selective inhibition index on COX-2, IC(50) (COX-1)/IC(50) (COX-2), of Cu-Asp was 3.33+/-0.89, while that of Asp was 0.42+/-0.12. The results suggest that, unlike Asp, Cu-Asp is a relatively selective inhibitor of COX-2 in the present models; the selectivity of Cu-Asp is about seven-fold greater than that of Asp.

Protection of preconditioning, postconditioning and combined therapy against hepatic ischemia/reperfusion injury.

OBJECTIVE: To study the protective effect of ischemic preconditioning (I-pre) and ischemic postconditioning (I-post) against ischemia/reperfusion (I/R) injury in rat's liver. METHODS: Using rat model of hepatic segmental I/R injury, rats were divided into 5 groups: Group A (sham group), Group B (I/R injury), Group C (I-pre group), Group D (I-post group) and Group E (combined treatment of I-pre and I-post). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and myeloperoxidase (MPO) in hepatic tissues were determined, respectively. In addition, 7 days'survival of Groups B, C, D and E were evaluated. RESULTS: Compared with Group B, Groups C, D and E exhibited significantly decreased ALT and AST release, minimized tissue injury, suppressed values of MDA and MPO, increased activities of SOD, GSH-Px and GSH (P less than 0.05), as well as improved animal survival. The differences among Groups C, D and E were not statistically significant. CONCLUSIONS: I-pre, I-post and combined therapy of I-pre and I-post have protective effect against hepatic I/R injury, which is correlated with its function of reducing the production of reactive oxygen species, maintaining the activities of antioxidant systems and suppressing neutrophils recruitment. No additive effect can be obtained in Group E.