N-octanoyl Dopamine Attenuates the Development of Transplant Vasculopathy in Rat Aortic Allografts Via Smooth Muscle Cell Protective Mechanisms.

BACKGROUND: Transplant vasculopathy (TV) is a major cause for late graft loss after cardiac transplantation. Endothelial damage and T cell infiltration play a pivotal role in the development of TV. Because N-octanoyl dopamine (NOD) inhibits vascular inflammation and suppresses T cell activation in vitro, we here tested the hypothesis that NOD treatment ameliorates TV. METHODS: Aortic grafts were orthotopically transplanted in the Dark Agouti to Brown Norway strain combination. Recipient rats were treated with NOD or vehicle administered via osmotic minipumps. Histology and quantitative polymerase chain reaction (qPCR) were performed on nontransplanted aortas and grafts explanted 2 and 4 weeks after transplantation to assess the degree of TV, inflammation, apoptosis, and number of (proliferating) α smooth muscle actin (αSMA) neointimal cells. In vitro analyses of human aortic smooth muscle cells were performed to test the effect of NOD on proliferation (WST-1 assay), cell cycle (flow cytometry and qPCR), and cytokine-induced apoptosis (flow cytometry). RESULTS: Allografts from vehicle-treated recipients developed neointimal lesions predominantly consisting of αSMA-expressing cells. NOD treatment significantly reduced neointima formation and neointimal αSMA cells. In situ, smooth muscle cell proliferation (Ki67) was not influenced by NOD. Macrophage (CD68), T (CD3), and Natural Killer (ANK61) cell infiltration as well as intragraft TNFα and IFNγ mRNA expression were similar in both groups. Medial apoptosis (cleaved caspase-3) was significantly reduced by NOD. In vitro, NOD inhibited proliferation of human aortic smooth muscle cells by causing a G1-arrest and protected from TNFα-induced apoptosis. CONCLUSIONS: This study identified NOD as potential treatment modality to attenuate TV. Our data clearly support a vasculoprotective effect of NOD by reducing smooth muscle cell proliferation and inflammation-induced apoptosis.

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.

Correlation of microRNA-16, microRNA-21 and microRNA-101 expression with cyclooxygenase-2 expression and angiogenic factors in cirrhotic and noncirrhotic human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression. METHODS: Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue. RESULTS: COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma. CONCLUSIONS: In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.

Smoking during pregnancy influences the maternal immune response in mice and humans.

OBJECTIVE: During pregnancy the maternal immune system has to adapt its response to accommodate the fetus. The objective of this study was to analyze the effects of smoking on the maternal immune system. STUDY DESIGN: First-trimester decidual tissue and peripheral blood of smoking and nonsmoking women were analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. A mouse model was used to further analyze the effects of smoking. Murine tissue was analyzed by flow cytometry, real-time RT-PCR, and immunohistochemistry. RESULTS: Smoking caused lower percentages of viable pups in mice and lower birthweights in humans. Smoking mothers, both mice and human, had more natural killer cells and inflammatory macrophages locally, whereas systemically they had lower percentages of regulatory T cells than nonsmoking controls. CONCLUSION: Maternal smoke exposure during pregnancy influences local and systemic immune responses in both women and mice. Such changes may be involved in adverse pregnancy outcomes in smoking individuals.

Renal effects of long-term darbepoetin alpha treatment in hypertensive TGR(mRen2)27 rats.

INTRODUCTION: Erytropoietin (EPO) has cytoprotective and angiogenic properties and has a beneficial effect in ischaemic conditions. Since the development of renal interstitial abnormalities are often associated with ischaemia, we studied the effects of the long-acting EPO analogue darbepoetin alpha (DA) on kidney damage in TGR(mRen2)27 (Ren2) rats. MATERIALS AND METHODS: Ren2 rats were randomised to DA or vehicle (VEH) or to DA + angiotensin converting enzyme inhibitor (ACEi) or VEH + ACEi. Sprague Dawley (SD) rats served as controls. Blood pressure was measured weekly and 24-h urine was collected to measure proteinuria. Blood samples were collected for creatinine and haematocrit. Kidneys were studied for inflammation and pre-fibrosis. Renal mRNA expression was studied for EPO, EPO-receptor, collagen-3α1 and kidney injury molecule-1 (KIM-1). RESULTS: DA had no effect on SBP, serum creatinine and proteinuria. Interstitial and glomerular α-SMA expression was significantly increased in Ren2. ACEi but not DA improved the increased renal inflammatory and pro-fibrotic profile in Ren2 rats. DA on top of ACEi further reduced glomerular α-SMA and KIM-1 expression. CONCLUSION: Long-term DA treatment has no beneficial effects on renal structural and functional changes in TGR(mRen2)27 rats in the time frame studied and the dose provided.

Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma.

BACKGROUND: Flat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA expression changes associated with invasive breast cancer, like miR-21, have not been studied in FEA. METHODS: We performed miRNA in-situ hybridization (ISH) on 15 cases with simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was investigated by immunohistochemistry. RESULTS: Two out of fifteen cases showed positive staining for miR-21 in normal breast ductal epithelium, seven out of fifteen cases were positive in the FEA component and nine out of twelve cases were positive in the DCIS component. A positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all three components were present in one tissue block and an increase of miR-21 from normal breast to FEA and to DCIS was observed in five cases. In three cases the FEA component was negative, whereas the DCIS component was positive for miR-21. In three other cases, normal, FEA and DCIS components were negative for miR-21 and in the last case all three components were positive. Overall we observed a gradual increase in percentage of miR-21 positive cases from normal, to FEA, DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1 staining decreased from positive in normal breast to negative in most DCIS and IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast tissue. CONCLUSION: Upregulation of miR-21 from normal ductal epithelial cells of the breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No clear relation was observed between the staining pattern of miR-21 and its previously reported target genes.

Enhanced ecto-apyrase activity of stimulated endothelial or mesangial cells is downregulated by glucocorticoids in vitro.

Endothelial as well as mesangial cells show enhanced activity of ecto-apyrase following pro-inflammatory stimulation in vitro. Since this ecto-enzyme appears to be able to regulate plasma hemopexin, which latter molecule plays a role in the pathogenesis of corticosteroid responsive nephrotic syndrome, the question was raised whether glucocorticoids are potentially able to downregulate ecto-apyrase activity of these cells. Therefore, cell cultures of endothelial or mesangial were stimulated with or without lipopolysaccharide (10 ng/ml). Parallel cultures were supplemented with prednisolone with or without the glucocorticoid receptor antagonist mifepristone in various concentrations. After 24 h, cytospins were prepared and cytochemically stained for ecto-apyrase activity. mRNA for apyrase of these cells was detected using reverse transcription-polymerase chain reaction (RT-PCR). Apyrase activity of either cells or soluble apyrase (0.16 U/ml buffer) with or without supplementation of prednisolone were biochemically assayed for their phosphatase activity. The results show significantly decreased ecto-apyrase activity of lipopolysaccharide-stimulated cells after treatment with prednisolone as compared to non-prednisolone-treated cells. Preincubation with mifepristone did not inhibit the effect of prednisolone. Identical mRNA signals for apyrase were found in prednisolone and non-prednisolone-treated cells. Interestingly, soluble apyrase also showed a significant decrease of activity following preincubation with prednisolone. It is concluded that prednisolone is able to downregulate ecto-apyrase of stimulated endothelial or mesangial cells, which may potentially inhibit the conversion of hemopexin to its pro-inflammatory isoform. As blocking of the cytosolic glucocorticoid receptor showed no effect upon the prednisolone action, whereas prednisolone is able to affect soluble apyrase per se, it is felt that this particular action of prednisolone may (at least partly) be mediated through a non-genomic pathway.

Specific MAP-kinase blockade protects against renal damage in homozygous TGR(mRen2)27 rats.

Angiotensin II (AngII) plays an important role in renal damage by acting on hemodynamics, cell-growth, proliferation, and fibrosis, mainly by effects on the AngII type 1 (AT(1)) receptor. The AT(1) receptor activates several intracellular signaling molecules such as mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and p38, but their role in AngII-mediated renal damage is not well characterized. We therefore investigated whether pharmacologic blockade of ERK and p38 could prevent renal damage in high-renin homozygous transgenic rats (Ren2), with the effects of an AT(1) receptor antagonist (AT(1)-RA) as a reference. Seven-week-old homozygous Ren2 rats were treated with low-dose AT(1)-RA candesartan, ERK inhibitor tyrphostin, or p38 inhibitor SB239063 for 4 weeks. Untreated Ren2 and SD rats served as controls. Blood pressure was measured at 7 and 11 weeks. At 11 weeks, plasma renin activity (PRA) and serum aldosterone were determined, and the animals were killed. Kidney sections were scored for glomerular and interstitial smooth muscle actin and glomerular desmin expression as early markers for renal damage. Mesangial matrix expansion was determined as a marker for structural damage. PRA and aldosterone levels were elevated in untreated Ren2 rats in comparison to SD controls. AT(1)-RA further increased PRA but decreased aldosterone. All parameters of renal damage were elevated in untreated Ren2 rats. Blood pressure was not elevated at week 7 in Ren2 and not affected by either treatment. Mild signs of hypertensive damage were found in untreated Ren2 rats. All interventions significantly diminished damage to glomerular epithelium and interstitium. In addition, AT(1) receptor and p38 blockade reduced mesangial matrix expansion. In homozygous Ren2 rats, renal damage was ameliorated by a nonhypotensive dose of an AT(1)-RA and, similarly, by blockade of ERK or p38. This suggests that ERK and p38 are involved in AngII-mediated renal damage.