Pacing-induced regional differences in adenosine receptors mRNA expression in a Swine model of dilated cardiomyopathy.

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). The aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n = 10) and from a control group (C, n = 4). ARs and TNF-α mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A1R (A1R: C = 0.6±0.2, PS = 0.1±0.04, OS = 0.04±0.01, p<0.0001 C vs. PS and OS respectively; A2AR: C = 1.04±0.59, PS = 2.62±0.79, OS = 2.99±0.79; A2BR: C = 1.2±0.1, PS = 5.59±2.3, OS = 1.59±0.46; A3R: C = 0.76±0.18, PS = 8.40±3.38, OS = 4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF-α mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction

Immunohistochemical localization of histidine-rich glycoprotein in human skeletal muscle: preferential distribution of the protein at the sarcomeric I-band

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration

Pacing-induced regional differences in adenosine receptors mRNA expression in a swine model of dilated cardiomyopathy

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). Aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n=10) and from a control group (C, n=4). ARs and TNF– mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A1R (A1R: C=0.6±0.2, PS=0.1±0.04, OS=0.04±0.01, p<0.0001 C vs. PS and OS respectively; A2AR: C=1.04±0.59, PS=2.62±0.79, OS=2.99±0.79; A2BR: C=1.2±0.1, PS=5.59±2.3, OS=1.59±0.46; A3R: C=0.76±0.18, PS=8.40±3.38, OS=4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF- mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction

Adenosine receptor expression in an experimental animal model of myocardial infarction with preserved left ventricular ejection fraction.

Adenosine, a purine nucleoside and a "retaliatory metabolite" in ischemia, is ubiquitous in the body and increases 100-fold during ischemia. Its biological actions are mediated by four adenosine receptors (ARs): A(1), A(2A), A(2B) and A(3). The aim of this study was to determine possible myocardial alterations in AR expression in an experimental animal model of myocardial infarction (MI) with a preserved left ventricular (LV) ejection fraction. LV tissue was collected from sexually mature male farm pigs with MI (n = 6) induced by permanent surgical ligation of the left anterior descending coronary artery and from five healthy pigs (C). mRNA expression of A(1)R, A(2A)R, A(2B)R, A(3)R and TNF-? was determined by real-time PCR in tissue collected from border (BZ) and remote zones (RZ) of the infarcted area and from LV of C. BZ, RZ and samples of C were stained immunohistochemically to investigate A(3)R immunoreaction. After 4 weeks a different regulation of ARs was observed. A(1)R mRNA expression was significantly lower in the infarct regions than in controls (C = 0.75 ? 0.2, BZ = 0.05 ? 0.2, RZ = 0.07 ? 0.02 p = 0.0025, p = 0.0016, C vs. BZ and RZ, respectively). Conversely A(3)R was higher in infarct areas (C = 0.94 ? 0.2, BZ = 2.85 ? 0.5, RZ = 3.48 ? 1.0, p = 0.048 C vs. RZ). No significant differences were observed for A(2A)R (C = 1.58 ? 0.6, BZ = 0.42 ? 0.1, RZ = 1.37 ? 0.6) and A(2B)R (C = 1.66 ? 0.2, BZ = 1.54 ? 0.5, RZ = 1.25 ? 0.4). A(3)R expression was confirmed by immunohistochemical analysis and was principally localized in cardiomyocytes. TNF-? mRNA results were: C 0.41 ? 0.25; BZ 1.60 ? 0.19; RZ 0.17 ? 0.04. The balance between A(1)R and A(3)R as well as between A(2A)R and A(2B)R was consistent with adaptative retaliatory anti-ischemic adenosinergic changes in the infarcted heart with preserved LV function

Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment

Aim: Glioblastoma multiforme is one of the deadliest forms of cancer, and current treatments are limited to palliative cares. The present study proposes a nanotechnology-based solution able to improve both drug efficacy and its delivery efficiency. Materials &amp; methods: Nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained nanovectors (nutlin-loaded magnetic solid lipid nanoparticle [Nut-Mag-SLNs]) were characterized by analyzing both their physicochemical properties and their effects on U-87 MG glioblastoma cells. Results: Nut-Mag-SLNs showed good colloidal stability, the ability to cross an in vitro blood–brain barrier model, and a superior pro-apoptotic activity toward glioblastoma cells with respect to the free drug. Conclusion: Nut-Mag-SLNs represent a promising multifunctional nanoplatform for the treatment of glioblastoma multiforme

High concentration of C-type natriuretic peptide promotes VEGF-dependent vasculogenesis in the remodeled region of infarcted swine heart with preserved left ventricular ejection fraction.

Abstract BACKGROUND: Vasculogenesis is a hallmark of myocardial restoration. Post-ischemic late remodeling is associated with pathology and function worsening. At the same time, neo-vasculogenesis helps function improving and requires the release of vascular endothelial growth factor type A (VEGF-A). The vasculogenic role of C-type natriuretic peptide (CNP), a cardiac paracrine hormone, is unknown in infarcted hearts with preserved left ventricular (LV) ejection fraction (EF). We explored whether myocardial VEGF-dependent vasculogenesis is affected by CNP. METHODS AND RESULTS: To this end, infarcted swine hearts were investigated by magnetic resonance imaging (MRI), histological and molecular assays. At the fourth week, MRI showed that transmural myocardial infarction (MI) affected approximately 13% of the LV wall mass without impairing global function (LVEF>50%, n=9). Increased fibrosis, metalloproteases and capillary density were localized to the infarct border zone (BZ), and were associated with increased expression of CNP (p=0.03 vs. remote zone (RZ)), VEGF-A (p<0.001 vs. RZ), BNP, a marker of myocardial dysfunction (p<0.01 vs. RZ) and the endothelial marker, factor VIII-related antigen (p<0.01 vs. RZ). In vitro, CNP 1000 nM promoted VEGF-dependent vasculogenesis without affecting the cell growth and survival, although CNP 100 nM or a high concentration of VEGF-A halted vascular growth. CONCLUSIONS: CNP expression is locally increased in infarct remodeled myocardium in the presence of dense capillary network. The vasculogenic response requires the co-exposure to high concentration of CNP and VEGF-A. Our data will be helpful to develop combined myocardial delivery of CNP and VEGF-A genes in order to reverse the remodeling process

Effect of dipyridamole on gap junctions regulation in diseased myocardium

Gap junctions (GJ) mediate electrical coupling between cardiac myocytes, allowing the spreading of the electrical wave responsible for synchronized contraction [1]. GJ function can be regulated by modulation of connexon densities on membranes, connexin (Cx) phosphorylation, trafficking and degradation. Recent studies showed that adenosine involves Cx43 turn-over in A1 receptor-dependent manner [2], and dipyridamole increases GJ coupling and amount of Cx43 in endothelial cells [3]. As the abnormalities in GJ organization and regulation have been implicated in diseased myocardium [1], the aim of the present study was to assess the regional expression of molecules involved in GJ regulation in a model of left ventricular disfunction (LVD). For this purpose the distribution and quantitative expression of Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, RhoA and A receptors, were investigated in experimental models of right ventricular-pacing induced LVD, undergoing concomitant dipyridamole therapy or placebo, and compared with healty myocardium obtained from sham operated minipigs. Results demonstrates that an altered pattern of factors involved in Cx43-made GJ regulation is present in ventricular myocardium with left ventricular dysfunction. Moreover, the dipyridamole treatment, that results in an improvement of heart function, seems to act also modulating expression and activation of these factors

Dipyridamole increases Cx43 expression in heart muscle cells through Adenosine 2A receptor/PKC pathway

Cx43, a predominant connexin in the heart, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and hemichannels that represent a pathway for the exchange of ions and metabolites between cytoplasm and the extracellular milieu. Our recent results (1) demonstrated that an altered distribution and quantitative expression of factors involved in Cx43-made GJ regulation as Cx43, its phosphorylated form pS368-Cx43, PKC phosphorylated substrates, and adenosine 2A receptor (A2AR) are present in ventricular myocardium with left ventricular dysfunction. Moreover, dipyridamole treatment, which shows a mild protective role on left ventricular function, seems to act through re-modulating the expression and activation of these factors. The role of these factors on signal transduction cascade triggered by dipyridamole was evaluated in this study by pharmacological and immunoistochemical experiments using the rat cardiomyoblast cell line H9c2. The treatment of H9c2 cells with dipyridamole enhanced the expression of Cx43, A2AR and PKC activity while induced a decrease of pS368-Cx43. Interestingly, we found that the A2AR activation was a prerequisite for the effects of dipyridamole, in fact, the pre-treatment with CSC, a selective A2AR receptor antagonist, abolished its effects on the expression of these factors

Cx43, RhoA and c-kit in diverticular disease

Diverticular disease (DD) is one of the most common diseases related to the gastrointestinal tract in Western countries. It has been postulated that abnormal colonic motility including increased overall motility, abnormal response to a physiologic stimulus and retropropagation of mass movement, predisposes to the formation of pulsion diverticula in the segments of the bowel containing the diverticula. Intestinal motility is regulated by complex interactions among smooth muscle cells (SMCs) of muscularis propria, enteric nerve endings and interstitial cells of Cajal (ICC). ICC are emerging as potential colonic pacemaker cells which modulate neuroenteric transmission by connecting SMC with varicosities of myenteric neuron axons. In colonic SMCs, transmenbrane channels rich in connexin (Cx), in particular Cx43, contribute to intercellular gap junctions, which ensure coordinated motor responses to nerve inputs.There is evidence that SMCs motility and gap junction permeability are regulated by the GTPase RhoA, an emerging key modulator of SMCs phenotype. The aim of the present study was to evaluate the Cx43 and RhoA expression in SMCs, as well as ICC density in colonic specimens from patients affected by diverticular disease (DD). Immunohistochemistry for Cx43, RhoA and c-kit and image analysis were used to examine muscularis propria of surgical whole thickness colonic samples from DD patients. Semiquantitative analysis of DD colonic specimens displayed a marked decrease in Cx43 and RhoA expression in SMCs. There was also a reduced ICC density in myenteric ganglia (ICC-MY), circular (ICC-CM) and longitudinal (ICC-LM) muscle, as compared to controls. Overall, it is suggested that abnormalities in gap junctions and RhoA expression in SMCs, together with a reduced density of muscular ICC, account for the colonic dismotility occurring in DD

Immunohistochemical Analysis of Myenteric Ganglia and Interstitial Cells of Cajal in Ulcerative Colitis

Ulcerative colitis (UC) is an inflammatory bowel disease with alterations of colonic motility, which influence clinical symptoms. Although morpho-functional abnormalities in the enteric nervous system have been suggested, in UC patients scarce attention has been paid to possible changes in the cells that control colonic motility, including myenteric neurons, glial cells, and interstitial cells of Cajal (ICC). This study evaluated the neural-glial components of myenteric ganglia and ICC in the colonic neuromuscular compartment of UC patients by quantitative immunohistochemical analysis. Full-thickness archival samples of the left colon were collected from 10 patients with UC (5 M, 5 F; age range, 45-62 years) who underwent elective bowel resection. The colonic neuromuscular compartment was evaluated immunohistochemically in paraffin cross-sections. The distribution and number of neurons, glial cells and ICC were assessed by anti-HuC/D, -S100β and -c-Kit antibodies, respectively. Data were compared with findings on archival samples of normal left colon from 10 sex- and age-matched control patients, who underwent surgery for uncomplicated colon cancer. Compared to controls, patients with UC showed: (a) reduced density of myenteric HuC/D-positive neurons and S100β-positive glial cells, with a loss over 61% and 38%, respectively, and increased glial cell/neuron ratio; (b) ICC decrease in the whole neuromuscular compartment. The quantitative variations of myenteric neuro-glial cells and ICC indicate considerable alterations of the colonic neuromuscular compartment in the setting of mucosal inflammation associated with UC, and provide a morphological basis for better understanding the motor abnormalities often observed in UC patients