Leonardo Gaspa

Importance of Ezh2 polycomb protein in tumorigenesis process interfering with the pathway of growth suppressive key elements.

An understanding of the mechanisms that uncover the dynamic changes in the distribution of the chromatin modifying enzymes and regulatory proteins on their target loci could provide further insight into the phenomenon of malignant transformation. Based on the current available data, it seems more and more clear that an abnormal expression of Ezh2, a member of the Polycomb group (PcG) protein, may be involved in the tumorigenesis process, in addition, different studies identify Ezh2 as a potential marker that distinguish aggressive prostate and breast cancer from indolent one. Recent investigation show that ectopic expression of Ezh2 provides proliferative advantage to primary cells through interaction with the pathways of key elements that control cell growth arrest and differentiation, like members of the retinoblastoma (Rb) family. Here, we outline how these pathways converge and we review the recent advances on the molecular mechanisms that promote cell cycle progression through deregulation of Ezh2 protein level, providing novel links between cancer progression and chromatin remodeling machineries. J. Cell. Physiol. 214: 295–300, 2008.

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Angiotensin AT1 receptor signalling modulates reparative angiogenesis induced by limb ischaemia

The concept that angiotensin II exerts pro‐angiogenic activity is not universally accepted. We evaluated whether inhibition of the renin‐angiotensin system (RAS) would influence reparative angiogenesis in a murine model of limb ischaemia. Perfusion recovery following surgical removal of the left femoral artery was analysed by laser Doppler flowmetry in mice given the ACE inhibitor ramipril (1 mg kg−1 per day), the AT1 antagonist losartan (15 mg kg−1 per day), or vehicle. Muscular capillarity was examined at necroscopy. Ramipril‐induced effects were also studied under combined blockade of kinin B1 and B2 receptors. Furthermore, the effects of ischaemia on AT1 gene expression and ACE activity were determined. In untreated mice, muscular AT1a gene expression was transiently decreased early after induction of limb ischaemia, whereas AT1b mRNA was up‐regulated. ACE activity was reduced in ischaemic muscles at 1 and 3 days. Gene expression of AT1 isoforms as well as ACE activity returned to basal values by day 14. Spontaneous neovascularization allowed for complete perfusion recovery of the ischaemic limb after 21 days. Reparative angiogenesis was negatively influenced by either ramipril (P<0.02) or losartan (P<0.01), leading to delayed and impaired post‐ischaemic recovery (50 – 70% less compared with controls). Ramipril‐induced effects remained unaltered under kinin receptor blockade. The present study indicates that (a) expression of angiotensin II AT1 receptors and ACE activity are modulated by ischaemia, (b) ACE‐inhibition or AT1 antagonism impairs reparative angiogenesis, and (c) intact AT1 receptor signalling is essential for post‐ischaemic recovery. These results provide new insights into the role of the RAS in vascular biology and suggest cautionary use of ACE inhibitors and AT1 antagonists in patients at risk for developing peripheral ischaemia.

Akt downregulation by flavin oxidase–induced ROS generation mediates dose-dependent endothelial cell damage elicited by natural antioxidants

High intake of natural antioxidants (NA) from plant-derived foods and beverages is thought to provide cardiovascular benefits. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from NA actions on endothelial cells (ECs) are actively investigated. Here, we show the direct impact of two NA, coumaric acid and resveratrol, on intracellular reactive oxygen species levels, protein carbonylation, and cell physiology in human ECs. While at lower doses, both NA promoted antioxidant effects, at moderately high doses, NA elicited a dose-dependent pro-oxidant effect, which was followed by apoptosis, cell damage, and phospho-Akt downregulation. NA-induced pro-oxidant effects were counteracted by N-acetyl cysteine and diphenyleneiodonium (DPI), suggesting a role for flavin oxidases in NA-induced toxicity. DPI also prevented NA-induced phospho-Akt downregulation indicating that Akt can work downstream of flavin oxidases in mediating cellular responses to NA. Stimulation of phospho-Akt by insulin dramatically counteracted NA-induced cell death, an effect abolished by Akt inhibition further suggesting that mechanistically Akt regulates cell survival in response to NA-induced stress. Although further studies are required to better characterize the molecular mechanism of NA-induced cell toxicity, our study is the first to show in a human vascular model that moderately high doses of NA can induce cell damage mediated by flavoproteins and the Akt pathway.

c-myc amplification in pre-malignant and malignant lesions induced in rat liver by the resistant hepatocyte model

We have investigated by restriction fragment analysis genomic abnormalities involving the c‐myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the “resistant hepatocyte” protocol in diethylnitrosamine‐initiated male F344 rats. Southern‐blot analysis of EcoRI‐restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c‐myc, in all tissues. c‐myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c‐myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII‐restricted DNA, and in HCCs by differential PCR. c‐myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13‐kb DNA extraband, hybridizing with c‐myc, was found in the HindIII‐restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1‐kb extra band was present in EcoRI‐digested DNA from one LA. EcoRI‐restricted DNA from some adenomas exhibited a decrease in intensity of the 18‐kb fragment, and an increase in intensity of the 3.2‐kb fragment. No alteration in banding pattern occurred in the β‐actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c‐myc gene, in pre‐malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c‐myc rearrangement in the progression of adenomas to malignancy.

Role of the bradykinin B2 receptor in the maturation of blood pressure phenotype: lesson from transgenic and knockout mice.

The binding of bradykinin (BK) to its B2 receptor results in a wide spectrum of biological effects including vasodilation, smooth muscle contraction and relaxation, pain, and inflammation. In order to gain a better insight into the physiological function of this potent vasoactive peptide, murine models have been created by the use of gene insertion or deletion. The results of studies using these strategies are revisited in the present article. In transgenic mice harboring the human BK B2 receptor cDNA (cHBKR), expression of the transgene was identified in the aorta, brain, heart, lung, liver, kidney, uterus and prostate gland by RT-PCR Southern blot analysis. These mice displayed an exaggerated hypotensive response to intra-aortic injection of BK, whereas the blood pressure of knockout mice, homozygous for targeted disruption of the endogenous gene, was insensitive to BK. Two transgenic mouse lines expressing the human BK B2 receptor showed a significant reduction of systolic tail-cuff blood pressure (84 +/- 1 mm Hg, n = 28; 80 +/- 1 mm Hg, n = 24; P < 0.001) compared with the control littermates (97 +/- 1 mm Hg, n = 52). Systolic blood pressure was elevated in BK B2 receptor knockout mice (124 +/- 1 mm Hg, n = 38). In heterozygous mice, systolic blood pressure was similar to that of controls until 5 month-old, then it raised to the elevated levels of knockout mice at 7 months of age. Together these data indicate that kinins acting through the B2 receptor play a role in the development of the blood pressure phenotype.

Opioid Peptide Gene Expression in the Primary Hereditary Cardiomyopathy of the Syrian Hamster I. REGULATION OF PRODYNORPHIN GENE EXPRESSION BY NUCLEAR PROTEIN KINASE C

Prodynorphin gene expression was investigated in adult ventricular myocytes isolated from normal (F1B) or cardiomyopathic (BIO 14.6) hamsters. Prodynorphin mRNA levels were higher in cardiomyopathic than in control myocytes and were stimulated by treatment of control cells with the protein kinase C (PKC) activator 1,2-dioctanoyl-sn-glycerol. Both chelerythrine and calphostin C, two PKC inhibitors, abolished the stimulatory effect of the diglyceride and significantly reduced prodynorphin gene expression in cardiomyopathic myocytes. Nuclear run-off experiments indicated that the prodynorphin gene was regulated at the transcriptional level and that treatment of nuclei isolated from control cells with 1,2-dioctanoyl-sn-glycerol increased prodynorphin gene transcription, whereas chelerythrine or calphostin C abolished this transcriptional effect. Direct exposure of nuclei isolated from cardiomyopathic myocytes to these inhibitors markedly down-regulated the rate of gene transcription. The expression of PKC-α, -δ, and -ε, as well as PKC activity, were increased in nuclei of cardiomyopathic myocytes compared with nuclei from control cells. The levels of both intracellular and secreted dynorphin B, a biologically active product of the gene, were higher in cardiomyopathic than in control cells and were stimulated or inhibited by cell treatment with 1,2-dioctanoyl-sn-glycerol or PKC inhibitors, respectively.

Albumin-bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS-PAGE, thiols were freed from protein with tri-n-butylphosphine and successively derivatized with 5-iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 microg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.

Role of calcitonin gene-related peptide and kinins in post-ischemic intestinal reperfusion

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.

Ramipril improves hemodynamic recovery but not microvascular response to ischemia in spontaneously hypertensive rats.

BACKGROUND Angiotensin converting enzyme (ACE) inhibition exerts positive effects on the microvasculature of normotensive animals, although this concept is not universally accepted. Recently, ACE inhibitors have been suggested to be useful for rescue in peripheral ischemia. METHODS We investigated whether chronic treatment with the ACE inhibitor ramipril may have a positive impact on the defective healing response to ischemia that is typical of spontaneously hypertensive rats (SHR). Unilateral limb ischemia was induced in 20-week-old SHR by surgically removing the left femoral artery. Rats were allowed to regain consciousness and then were randomly allocated to treatment with ramipril (1 mg/kg body weight in drinking water) or vehicle for 28 days. RESULTS The SHR failed to develop reparative angiogenesis in response to ischemia, thus having inadequate perfusion recovery. Ramipril reduced both tail-cuff systolic blood pressure (180 +/- 7 v 207 +/- 2 mm Hg in the vehicle group at 28 days, P < .05) and intra-arterial mean blood pressure (115 +/- 6 v 135 +/- 5 mm Hg in the vehicle group, P < .05). These effects were associated with increased responsiveness to endothelium-dependent vasodilatation by acetylcholine. Treatment with ramipril did not influence muscular capillary and arteriole density but accelerated the rate of perfusion recovery, leading to complete healing within 28 days after surgery. CONCLUSIONS These results indicate that ACE inhibition by ramipril may be useful for the treatment of peripheral vascular complications in hypertension.