Optimized detection and segmentation of nuclei in gastric cancer images using stain normalization and blurred artifact removal.

Histological analysis with microscopy is the gold standard to diagnose and stage cancer, where slides or whole slide images are analyzed for cell morphological and spatial features by pathologists. The nuclei of cancerous cells are characterized by nonuniform chromatin distribution, irregular shapes, and varying size. As nucleus area and shape alone carry prognostic value, detection and segmentation of nuclei are among the most important steps in disease grading. However, evaluation of nuclei is a laborious, time-consuming, and subjective process with large variation among pathologists. Recent advances in digital pathology have allowed significant applications in nuclei detection, segmentation, and classification, but automated image analysis is greatly affected by staining factors, scanner variability, and imaging artifacts, requiring robust image preprocessing, normalization, and segmentation methods for clinically satisfactory results. In this paper, we aimed to evaluate and compare the digital image analysis techniques used in clinical pathology and research in the setting of gastric cancer. A literature review was conducted to evaluate potential methods of improving nuclei detection. Digitized images of 35 patients from a retrospective cohort of gastric adenocarcinoma at Oulu University Hospital in 1987-2016 were annotated for nuclei (n = 9085) by expert pathologists and 14 images of different cancer types from public TCGA dataset with annotated nuclei (n = 7000) were used as a comparison to evaluate applicability in other cancer types. The detection and segmentation accuracy with the selected color normalization and stain separation techniques were compared between the methods. The extracted information can be supplemented by patient's medical data and fed to the existing statistical clinical tools or subjected to subsequent AI-assisted classification and prediction models. The performance of each method is evaluated by several metrics against the annotations done by expert pathologists. The F1-measure of 0.854 ± 0.068 is achieved with color normalization for the gastric cancer dataset, and 0.907 ± 0.044 with color deconvolution for the public dataset, showing comparable results to the earlier state-of-the-art works. The developed techniques serve as a basis for further research on application and interpretability of AI-assisted tools for gastric cancer diagnosis.

Menaquinone 4 increases plasma lipid levels in hypercholesterolemic mice.

In calcific aortic valve disease (CAVD) progressive valvular calcification causes aortic valve dysfunction. CAVD has several risk factors such as age and dyslipidemia. Vitamin K was shown to inhibit vascular calcification in mice and valvular calcification in patients with CAVD. We studied the effect of menaquinone 4 (MK4/vitamin K2) on valvular calcification in the hypercholesterolemic mouse model of CAVD. LDLr-/-ApoB100/100 male mice were fed with a Western diet for 5 months, with (n = 10) or without (n = 10) added 0.2 mg/g MK4. Body weight gain was followed weekly. Morphology of aortic valves and liver was assessed with immunohistochemistry. Plasma cholesterol levels and cytokines from hepatic tissue were assessed in the end of the study. Hepatic gene expression of lipid metabolism regulating genes were assessed after 18 h diet. MK4 exacerbated the lipoprotein lipid profile without affecting aortic valve morphology in hypercholesterolemic LDLr-/- ApoB100/100 mice. The MK4-containing WD diet increased plasma levels of LDL and triglycerides, hepatic steatosis, and mRNA expression of genes required for triglyceride and cholesterol synthesis. MK4 diminished levels of several cytokines and chemokines in liver, including IL-6, TNFα and MCP1, as measured by hepatic cytokine array. Consequently, MK4 may exert non-beneficial effects on circulating lipid levels, especially in hypercholesterolemic individuals.

Heat shock protein 90 is downregulated in calcific aortic valve disease.

BACKGROUND: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. METHODS: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. RESULTS: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. CONCLUSIONS: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.

Characterizing valve dynamics in mice by high-resolution cine-MRI.

AIMS: In calcific aortic valve disease (CAVD), progressive valvular sclerosis and calcification cause narrowing of the orifice and an impairment of the valve's function. We applied high-resolution cine-MRI to perform quantitative analysis of the dynamics of the aortic valve in a mice model of CAVD. METHODS AND RESULTS: LDLr-/- ApoB100/100 mice were fed a Western diet (WD) or a standard diet (control) for 22 weeks. The mice were imaged in a 7 T horizontal MRI scanner, and aortic valve dynamics was examined by imaging the cross-section of the aorta at valve level using cine sequences. From these images, the area of the aortic valve orifice was determined during the heart cycle. MRI results were compared with echocardiographic and histopathologic results. The data revealed evidence of clear aortic valve dysfunction in WD mice as compared with control mice (interaction P < 0.001). MRI showed narrowing (14%, P < 0.05) of the orifice area, and this was also seen in histology (34%, P < 0.05), indicating more severe aortic stenosis after WD than in controls. Additionally, MRI revealed a reduction in the ejection fraction (EF) (-11%, P < 0.01), a result confirmed with echocardiography (-27%, P < 0.001) in mice fed with WD. EF detected by MRI and echocardiography also correlated strongly with the degree of stenosis assessed by histology. CONCLUSIONS: Cine-MRI can be used for quantitative analysis of the aortic valve orifice over the cardiac cycle in mice. MRI showed the cusps clearly, and we were able to detect aortic valve dysfunction over time through the cardiac cycle.

Increased mesenchymal podoplanin expression is associated with calcification in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (AR + f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in AR + f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.

Nuclear factor E2-related factor 2 deficiency impairs atherosclerotic lesion development but promotes features of plaque instability in hypercholesterolaemic mice.

Aims: Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR-/-), and LDLR-/- mice expressing apoB-100 only (LDLR-/- ApoB100/100) having a humanized lipoprotein profile. Methods and results: LDLR-/- mice were fed a high-fat diet (HFD) for 6 or 12 weeks and LDLR-/-ApoB100/100 mice a regular chow diet for 6 or 12 months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR-/- mice reduced total plasma cholesterol after 6 weeks of HFD and triglycerides in LDLR-/-ApoB100/100 mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR-/-ApoB100/100 mice as it increased plaque calcification. Moreover, ∼36% of Nrf2-/-LDLR-/-ApoB100/100 females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12 months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR-/-ApoB100/100 female mice at age of 12 months. Conclusions: Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR-/-ApoB100/100 mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.

Mitogen-activated protein kinase p38 target regenerating islet-derived 3γ expression is upregulated in cardiac inflammatory response in the rat heart.

Regenerating islet-derived 3γ (Reg3γ) is a multifunctional protein, associated with various tissue injuries and inflammatory states. Since chronic inflammation is characteristics also for heart failure, the aim of this study was to characterize Reg3γ expression in cardiac inflammatory conditions. Reg3γ expression was studied in experimental rat models of myocardial infarction (MI) and pressure overload in vivo. For cell culture studies neonatal rat cardiac myocytes (NRCMs) were used. In addition, adenovirus-mediated gene transfer of upstream mitogen-activated protein kinase (MAPK) kinase 3b and p38α MAPK in vivo and in vitro was performed. Reg3γ mRNA (12.8-fold, P < 0.01) and protein (5.8-fold, P < 0.001) levels were upregulated during the postinfarction remodeling at day 1 after MI, and angiotensin II (Ang II) markedly increased Reg3γ mRNA levels from 6 h to 2 weeks. Immunohistochemistry revealed that the Ang II-induced expression of Reg3γ was localized into the cardiac fibroblasts and myofibroblasts of the proliferating connective tissue in the heart. Stretching and treatments with endothelin-1, lipopolysaccharide (LPS), and fibroblast growth factor-1 increased Reg3γ mRNA levels in NRCMs. SB203580, a selective p38 MAPK inhibitor, markedly attenuated LPS and mechanical stretch-induced upregulation of Reg3γ gene expression. Moreover, combined overexpression of MKK3bE and WT p38α increased Reg3γ gene expression in cultured cardiomyocytes in vitro and in the rat heart in vivo. Our study shows that cardiac stress activates Reg3γ expression and p38 MAPK is an upstream regulator of Reg3γ gene expression in heart. Altogether our data suggest Reg3γ is associated with cardiac inflammatory signaling.

Distal Inside-Out Epineural Sliding Technique to Repair Segmental Nerve Defects.

Background: The repair of a segmental peripheral nerve injury is a clinical challenge. Several studies have been performed to determine superior methods for overcoming nerve gaps. The purpose of this study was to investigate if the inside-out slided epineurium of the distal segment of an injured nerve can serve as a conduit to bridge a short nerve defect (10 mm). Methods: Nineteen sciatic nerves in Sprague-Dawley rats were transected, and a 10-mm gap was left between the ends. A section of distal epineurium was pulled inside out to bridge the gap. Walking track analysis was performed, and the sciatic function index (SFI) was calculated. Wet muscle mass and withdrawal reflex were measured. The density of axon fibers at different levels of repaired nerves was determined, and histological analysis was performed at 16 weeks. Results: The mean SFI improved from -81.0 at 4 weeks to 36.3 at 16 weeks. The axon densities showed regeneration through the epineural tube, and 5 of the rats demonstrated a withdrawal reflex. The weight of the tibialis anterior muscle of the injured limb at 16 weeks was 59% that of the uninjured side. Conclusions: The distal epineural sheath tube provided a size-matched conduit between the nerve stumps, with no histological donor-site morbidity. Histologically, regeneration occurred through the epineural tube without neuroma formation, and functional recovery was comparable to that of previous studies of nerve repair techniques. Technique may be an addition to the armamentarium of tools used to treat segmental nerve defects.

TSC-22 up-regulates collagen 3a1 gene expression in the rat heart.

BACKGROUND: The transforming growth factor (TGF)-ß is one of the key mediators in cardiac remodelling occurring after myocardial infarction (MI) and in hypertensive heart disease. The TGF-ß-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains unknown. METHODS: The aim of the present study was to characterize cardiac TSC-22 expression in vivo in cardiac remodelling and in myocytes in vitro. In addition, we used TSC-22 gene transfer in order to examine the effects of TSC-22 on cardiac gene expression and function. RESULTS: We found that TSC-22 is rapidly up-regulated by multiple hypertrophic stimuli, and in post-MI remodelling both TSC-22 mRNA and protein levels were up-regulated (4.1-fold, P <0.001 and 3.0-fold, P <0.05, respectively) already on day 1. We observed that both losartan and metoprolol treatments reduced left ventricular TSC-22 gene expression. Finally, TSC-22 overexpression by local intramyocardial adenovirus-mediated gene delivery showed that TSC-22 appears to have a role in regulating collagen type IIIα1 gene expression in the heart. CONCLUSIONS: These results demonstrate that TSC-22 expression is induced in response to cardiac overload. Moreover, our data suggests that, by regulating collagen expression in the heart in vivo, TSC-22 could be a potential target for fibrosis-preventing therapies.

The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks' follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

WDR12, a Member of Nucleolar PeBoW-Complex, Is Up-Regulated in Failing Hearts and Causes Deterioration of Cardiac Function.

AIMS: In a recent genome-wide association study, WD-repeat domain 12 (WDR12) was associated with early-onset myocardial infarction (MI). However, the function of WDR12 in the heart is unknown. METHODS AND RESULTS: We characterized cardiac expression of WDR12, used adenovirus-mediated WDR12 gene delivery to examine effects of WDR12 on left ventricular (LV) remodeling, and analyzed relationship between MI associated WDR12 allele and cardiac function in human subjects. LV WDR12 protein levels were increased in patients with dilated cardiomyopathy and rats post-infarction. In normal adult rat hearts, WDR12 gene delivery into the anterior wall of the LV decreased interventricular septum diastolic and systolic thickness and increased the diastolic and systolic diameters of the LV. Moreover, LV ejection fraction (9.1%, P<0.05) and fractional shortening (12.2%, P<0.05) were declined. The adverse effects of WDR12 gene delivery on cardiac function were associated with decreased cellular proliferation, activation of p38 mitogen-activated protein kinase (MAPK)/heat shock protein (HSP) 27 pathway, and increased protein levels of Block of proliferation 1 (BOP1), essential for ribosome biogenesis. Post-infarction WDR12 gene delivery decreased E/A ratio (32%, P<0.05) suggesting worsening of diastolic function. In human subjects, MI associated WDR12 allele was associated significantly with diastolic dysfunction and left atrial size. CONCLUSIONS: WDR12 triggers distinct deterioration of cardiac function in adult rat heart and the MI associated WDR12 variant is associated with diastolic dysfunction in human subjects.

(Pro)renin receptor triggers distinct angiotensin II-independent extracellular matrix remodeling and deterioration of cardiac function.

BACKGROUND: Activation of the renin-angiotensin-system (RAS) plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (pro)renin receptor ((P)RR) is not yet solved. We determined here the direct functional and structural effects of (P)RR in the heart. METHODOLOGY/PRINCIPAL FINDINGS: (P)RR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (P)RR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01), fractional shortening (P<0.01), and intraventricular septum diastolic and systolic thickness, associated with approximately 2-fold increase in left ventricular (P)RR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (P)RR gene overexpression was mediated by angiotensin II (Ang II), we infused an AT(1) receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (P)RR overexpressing animals as well. Intramyocardial (P)RR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-ß1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (P)RR gene delivery was Ang II-dependent. Finally, (P)RR overexpression significantly increased direct protein-protein interaction between (P)RR and promyelocytic zinc-finger protein. CONCLUSIONS/SIGNIFICANCE: These results indicate for the first time that (P)RR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (P)RR as a novel therapeutic target to optimize RAS blockade in failing hearts.

Increased thrombospondin-2 in human fibrosclerotic and stenotic aortic valves.

BACKGROUND: Active involvement of extracellular matrix (ECM) and its composition regulating factors may have a central role in the pathogenesis of calcific aortic valve disease (CAVD). Thrombospondins (TSPs) are highly conserved matricellular proteins regulating inflammation, angiogenesis and ECM remodeling. These processes are strongly associated with progression of aortic valve stenosis (AS). However, the expression of TSPs in CAVD is not known. METHODS: We characterized the expression of TSPs 1-4 in human aortic valves by real-time quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Control valves (n=8), thickened and stiffened fibro(sclero)tic valves (n=8), and calcified AS valves (n=24) were compared. Furthermore, potential factors regulating TSP-2 expression was studied by western blotting and gel mobility shift assay in another set of control (n=10) and AS (n=20) valves. RESULTS: TSP-2 mRNA levels were increased 4.9-fold (P=0.037) and 4.8-fold (P=0.001) in fibro(sclero)tic and stenotic valves, respectively, whereas the expression of other TSPs did not change significantly. All TSPs 1-4 were detected from aortic valves by immunohistochemistry. Positive TSP-2 immunostaining was seen in the valvular myofibroblasts and patchily in endothelial cells. Semiquantitative analysis of TSP-2 staining indicated increased immunoreactivity for TSP-2 in neo vessels of fibro(sclero)tic and calcified aortic valves. Finally, when compared to controls, AS was associated with significant down regulation of Akt-pathway and diminished binding activity of nuclear factor-κB (NF-κB). CONCLUSIONS: We report for the first time that TSPs 1-4 are expressed in human aortic valves. CAVD is characterized by myofibroblastic proliferation and neovascularization associated upregulation of TSP-2 expression, as well as inactivation of Akt and NF-κB.

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas--correlation with array comparative genome hybridization results and IDH1 mutation.

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

Left ventricular periostin gene expression is associated with fibrogenesis in experimental renal insufficiency.

BACKGROUND: Cardiovascular diseases are the most important cause of death in patients with impaired kidney function. Left ventricular hypertrophy (LVH), cardiac interstitial fibrosis and cardiovascular calcifications are characteristic of chronic renal insufficiency (CRI). Periostin is a fibrogenesis- and calcification-related matricellular protein re-expressed in adult tissues undergoing remodelling in response to pathological stimuli. The role of periostin in CRI-induced LVH is unknown. METHODS: Rats were 5/6-nephrectomized (NX), and after 15 weeks of disease progression high-calcium, high-phosphate or paricalcitol treatment was given for 12 weeks. Cardiac tissue and blood samples were taken to study periostin gene expression and to determine factors contributing to its reactivation, respectively. Left ventricular (LV) periostin expression was also examined in response to angiotensin II or arginine(8)-vasopressin (AVP)-induced pressure overload and in spontaneously hypertensive rats. RESULTS: CRI resulted in a 6.5-fold increase in LV periostin messenger RNA (mRNA) levels. Positive extracellular immunostaining for periostin was detected in areas of infiltrated inflammatory cells and fibrotic lesions. There was a significant correlation between LV periostin mRNA levels and plasma biomarkers of impaired kidney function, LVH, fibrogenesis-related proteins osteopontin and osteoactivin, and anti-calcific matrix Gla protein. Moreover, LV periostin gene expression in CRI correlated positively with systolic blood pressure (BP) and was activated rapidly in response to angiotensin II or AVP infusions. CONCLUSIONS: Periostin is involved in fibrotic cardiac remodelling in CRI. The re-expression of periostin is localized to the fibrotic and inflammatory lesions and is most likely the consequence of elevated BP.

Statin treatment and gene expression of anti-atherogenic factor C-type natriuretic peptide system in stenotic aortic valves.

AIMS: Aortic valve calcification is an actively regulated process with endothelial dysfunction displaying hallmarks of atherosclerosis. C-type natriuretic peptide (CNP) system has been reported to have a role in the pathogenesis of vascular atherosclerosis and to be distinctly downregulated in aortic valve stenosis (AS). Here we studied gene expressions of CNP and is target receptor natriuretic peptide receptor type B (NPR-B) in human aortic valves. Furthermore, we compared gene expression of CNP system in patients with HMG-coenzyme-A reductase (statin) treatment to non-statin-treated patients in AS group. METHODS AND RESULTS: With the study population of 108 patients, we characterized expression of CNP and NPR-B in human aortic valves and compared normal control valves (n = 12) with valves obtained from patients with aortic regurgitation (AR, n = 16), AR with fibrosis (AR+fibr., n = 19) and AS (n = 61). By reverse transcription-polymerase chain reaction (RT-PCR), CNP mRNA levels were 89% lower (p = 0.022) in stenotic valves, when compared to AR group. Moreover, the mRNA levels of NPR-B, the target receptor of CNP, were 62% lower (p < 0.001) in stenotic valves when compared to control group and 54% lower (p = 0.002) in stenotic valves, when compared to AR group. There was no statistical significant difference in CNP and NPR-B levels in AS group when the statin-treated patients were compared to untreated patients. CONCLUSIONS: These results show for the first time that the gene expression of anti-atherogenic CNP system did not differ between statin-treated and non-statin-treated patients in AS. The research data supports the results of clinical trials with the same drug class.

(Pro)renin receptors and angiotensin converting enzyme 2/angiotensin-(1-7)/Mas receptor axis in human aortic valve stenosis.

BACKGROUND: There is increasing evidence that renin-angiotensin system (RAS) may play a major role in the actively regulated fibrocalcific process in aortic valve stenosis (AS), but the gene expression or function of (pro)renin receptor ((P)RR), prorenin and renin or angiotensin converting enzyme 2(ACE2)/angiotensin-(1-7)/Mas receptor axis in calcific aortic valve disease is not known. METHODS AND RESULTS: We characterized expression of (P)RR, ACE2 and Mas receptor as well as renin, prorenin and angiotensin II type 2 (AT(2)) receptors in human aortic valves, and compared normal control valves (n = 11) with valves obtained from patients with aortic regurgitation (AR, n = 14), AR with fibrosis (n = 20) and AS (n = 61). By immunohistochemistry (P)RR positive staining was seen in the valvular endothelial cells of control and in the neovessels of stenotic valves. By RT-PCR, renin mRNA levels were 72% (P = 0.001) and prorenin mRNA levels 64% lower (P = 0.002) in stenotic aortic valves compared to control valves. ACE2, Mas receptor and AT(2)-receptor mRNA levels were 69% (P < 0.001), 58% (P = 0.008) and 75% (P = 0.001) lower, respectively, in stenotic valves. ACE2 positive staining, existing to lesser extent in stenotic aortic valves, was localized mainly to stromal area in spongiosa layer in control valves. CONCLUSIONS: (P)RR, prorenin and renin are expressed in human aortic valves. We also report for the first time expression of ACE2/angiotensin-(1-7)/-Mas receptor axis in human aortic valve cusps. The downregulation of ACE2/angiotensin-(1-7)/-Mas receptor axis as well as AT(2)-receptors may promote fibrosis, proliferation and inflammation in patients with AS.

Apelin and its receptor APJ in human aortic valve stenosis.

BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process that shows some hallmarks of atherosclerosis. Apelin and its receptor, APJ, are highly expressed in the heart, and the proposed effects of the apelin-APJ system are opposite to those of the angiotensin II-AT1-receptor pathway. The role of the apelin-APJ signaling pathway in calcified aortic valve disease is unknown. METHODS: The study involved the characterization and comparison of expression of apelin and APJ as well as angiotensin II receptors (AT1 and AT2) in the aortic valves of patients with normal valves (n = 6), aortic regurgitation (n = 9 AR), regurgitation and fibrosis/mild sclerosis (n = 14), and AS (n = 25). RESULTS: By employing the reverse-transcriptase polymerase chain reaction (RT-PCR), the gene expression of apelin (3.63-fold, p = 0.001) and the APJ receptor (2.70-fold, p = 0.01) were shown to be significantly up-regulated in stenotic valves when compared to controls. In addition, APJ receptor mRNA levels were higher (2.9-fold, p = 0.010) in the AR + sclerosis group when compared to controls. Using immunohistochemistry, apelin was shown to be localized in stenotic aortic valves to the valvular endothelial layer of the aortic valve, to vascular endothelial cells in neovessels, and to fibroblasts and macrophages adjacent to vessels in the stromal area. AT2-receptor mRNA levels were 90% (p < 0.001) lower in stenotic valves. In contrast, the gene expression of AT1-receptors did not differ significantly among the groups. CONCLUSION: Aortic valve stenosis is characterized by an up-regulation of the apelin-APJ signaling pathway, revealing a possible novel target for drug discovery in calcified aortic valve disease by suppressing chemotaxis, angiogenesis and osteoblast activity, all of which are well-documented phenomena in the disease process.

Budding invasive margin and prognosis in colorectal cancer--no direct association with beta-catenin expression.

Cancer cell budding at the invasive margin has been associated with poor prognosis in rectal cancer. beta-Catenin is an adhesion protein involved in the nuclear Wnt/beta-catenin pathway, and mesenchymal transition of colorectal cancer cells. Hence, we investigated the relationship between cancer cell budding at the invasive margin, beta-catenin expression, and 5-year-survival in colorectal cancer. Four hundred and sixty six colorectal cancer specimens were analysed for budding margin, and 108 specimens from the same set for beta-catenin by immunohistochemistry. A budding margin was present in 24.0% of the cases and predicted a poor 5-year-survival (15.4%, P < 0.00001). Nuclear beta-catenin expression increased from the central area towards the invasive margin (P < 0.001), but did not predict budding. Budding margin is an independent factor associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Nuclear beta-catenin was often found at the invasive margin, but is unlikely to be the sole cause of budding.

Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite.

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. Recent studies have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2) side chain. Alendronate, like other nitrogen-containing bisphosphonates, inhibits bone resorption and causes apoptosis of osteoclasts and other cells in vitro by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. Clodronate, a bisphosphonate that lacks a nitrogen, does not inhibit protein isoprenylation but can be metabolized intracellularly to a beta-gamma-methylene (AppCp-type) analog of ATP, which is cytotoxic to macrophages in vitro. The detailed molecular basis for the cytotoxic effects of adenosine-5'-[beta,gamma-dichloromethylene]triphosphate (AppCCl(2)p) has not been determined yet. We addressed this question by studying the effects of alendronate, clodronate, and the clodronate metabolite AppCCl(2)p on isolated mitochondria, mitochondrial fractions, and mitochondrial membrane potential in isolated human osteoclasts. We found that AppCCl(2)p inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase. Alendronate or the native form of clodronate did not have any immediate effect on mitochondria. However, longer treatment with liposome-encapsulated clodronate caused collapse of the mitochondrial membrane potential, although prominent apoptosis was a late event. Hence, inhibition of the ADP/ATP translocase by the metabolite AppCCl(2)p is a likely route by which clodronate causes osteoclast apoptosis and inhibits bone resorption.