Vincenzo Cimini

Ischemic Neoangiogenesis Enhanced by β2-Adrenergic Receptor Overexpression: A Novel Role for the Endothelial Adrenergic System

&bgr;2-Adrenergic receptors (&bgr;2ARs) are widely expressed, although their physiological relevance in many tissues is not yet fully understood. In vascular endothelial cells, they regulate NO release and vessel tone. Here we provide novel evidence that &bgr;2ARs can regulate neoangiogenesis in response to chronic ischemia. We used in vivo adenoviral-mediated gene transfer of the human &bgr;2AR to the endothelium of the rat femoral artery and increased &bgr;2AR signaling resulting in ameliorated angiographic blood flow and hindlimb perfusion after chronic ischemia. Histological analysis confirmed that &bgr;2AR overexpression also produced benefits on capillary density. The same maneuver partially rescued impaired angiogenesis in spontaneously hypertensive rats (SHR), whereas gene delivery of the G-protein–coupling defective mutant Ile164 &bgr;2AR failed to provide ameliorations. Stimulation of endogenous and overexpressed &bgr;2AR on endothelial cells in vitro was found to regulate cell number by inducing proliferation and [3H]-thymidine incorporation through means of extracellular receptor-activated kinase and vascular endothelial growth factor. The &bgr;2AR also has novel effects on endothelial cell number through stimulation of proapoptosis and antiapoptosis pathways involving p38 mitogen-activated protein kinase and PI3-kinase/Akt activation. Therefore, &bgr;2ARs play a critical role in endothelial cell proliferation and function including revascularization, suggesting a novel and physiologically relevant role in neoangiogenesis in response to ischemia.

The G-protein-coupled receptor kinase 5 inhibits NFkappaB transcriptional activity by inducing nuclear accumulation of IkappaB alpha.

G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5–IκBα interaction on NFκB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IκBα, leading to the inhibition of NFκB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IκBα, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IκBα as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFκB, we evaluated the effects of GRK5-RH on NFκB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFκB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFκB activity.

In vivo properties of the proangiogenic peptide QK

The main regulator of neovascularization is Vascular Endothelial Growth Factor (VEGF). We recently demonstrated that QK, a de novo engineered VEGF mimicking peptide, shares in vitro the same biological properties of VEGF, inducing capillary formation and organization. On these grounds, the aim of this study is to evaluate in vivo the effects of this small peptide. Therefore, on Wistar Kyoto rats, we evaluated vasomotor responses to VEGF and QK in common carotid rings. Also, we assessed the effects of QK in three different models of angiogenesis: ischemic hindlimb, wound healing and Matrigel plugs. QK and VEGF present similar endothelium-dependent vasodilatation. Moreover, the ability of QK to induce neovascularization was confirmed us by digital angiographies, dyed beads dilution and histological analysis in the ischemic hindlimb as well as by histology in wounds and Matrigel plugs. Our findings show the proangiogenic properties of QK, suggesting that also in vivo this peptide resembles the full VEGF protein. These data open to new fields of investigation on the mechanisms of activation of VEGF receptors, offering clinical implications for treatment of pathophysiological conditions such as chronic ischemia.

β2-Adrenergic Receptor Gene Delivery to the Endothelium Corrects Impaired Adrenergic Vasorelaxation in Hypertension

Background—Impaired &bgr;-adrenergic receptor (AR)–mediated vasorelaxation in hypertension plays a role in increased peripheral vascular resistance and blood pressure. Because the &bgr;2AR is the most abundant vascular AR subtype, we sought to enhance &bgr;AR vasorelaxation by overexpressing &bgr;2ARs via adenoviral-mediated gene transfer (AD&bgr;2AR) to the vascular endothelium of the carotid artery. Methods and Results—In normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, we exposed the right common carotid artery to AD&bgr;2AR in situ for 15 minutes by injection into the lumen while the blood flow was interrupted. Control carotids received an empty vector (ADempty). Three days later, transgene expression and selective endothelial localization were confirmed in infected vessels. Vasoregulation after &bgr;2AR overexpression (2-fold) was studied in isolated organ baths. AD&bgr;2AR carotid responses to &agr;1AR and &agr;2AR agonists were not affected, whereas responses to epinephrine were altered and &bgr;AR-mediated vasorelaxation was enhanced after &bgr;2AR overexpression. As expected, &bgr;AR-mediated vasodilatation in control carotids of SHR rats was significantly less than in similar control WKY carotid arteries. AD&bgr;2AR treatment enhanced &bgr;AR vasorelaxation in SHR to levels similar to those seen in AD&bgr;2AR WKY carotids. Conclusions—Our results demonstrate a critical role for the endothelium in &bgr;AR-mediated vasorelaxation and suggest that impaired &bgr;AR signaling may account for dysfunctional &bgr;AR vasorelaxation in hypertension rather than impaired endothelium-dependent nitric oxide metabolism.

Endothelial α1-adrenoceptors regulate neo-angiogenesis

Intact endothelium plays a pivotal role in post‐ischaemic angiogenesis. It is a phenomenon finely tuned by activation and inhibition of several endothelial receptors. The presence of α1‐adrenoceptors on the endothelium suggests that these receptors may participate in regenerative phenomena by regulating the responses of endothelial cells involved in neo‐angiogenesis.

Myocardial β2-adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure

BACKGROUND AND PURPOSE We investigated whether β2‐adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart.

Genetic Deletion of Uncoupling Protein 3 Exaggerates Apoptotic Cell Death in the Ischemic Heart Leading to Heart Failure

Background Uncoupling protein 3 (ucp3) is a member of the mitochondrial anion carrier superfamily of proteins uncoupling mitochondrial respiration. In this study, we investigated the effects of ucp3 genetic deletion on mitochondrial function and cell survival under low oxygen conditions in vitro and in vivo. Methods and Results To test the effects of ucp3 deletion in vitro, murine embryonic fibroblasts and adult cardiomyocytes were isolated from wild‐type (WT, n=67) and ucp3 knockout mice (ucp3−/−, n=70). To test the effects of ucp3 genetic deletion in vivo, myocardial infarction (MI) was induced by permanent coronary artery ligation in WT and ucp3−/− mice. Compared with WT, ucp3−/− murine embryonic fibroblasts and cardiomyocytes exhibited mitochondrial dysfunction and increased mitochondrial reactive oxygen species generation and apoptotic cell death under hypoxic conditions in vitro (terminal deoxynucleotidyl transferase‐dUTP nick end labeling–positive nuclei: WT hypoxia, 70.3±1.2%; ucp3−/− hypoxia, 85.3±0.9%; P<0.05). After MI, despite similar areas at risk in the 2 groups, ucp3−/− hearts demonstrated a significantly larger infarct size compared with WT (infarct area/area at risk: WT, 48.2±3.7%; ucp3−/−, 65.0±2.9%; P<0.05). Eight weeks after MI, cardiac function was significantly decreased in ucp3−/− mice compared with WT (fractional shortening: WT MI, 42.7±3.1%; ucp3−/− MI, 24.4±2.9; P<0.05), and this was associated with heightened apoptotic cell death (terminal deoxynucleotidyl transferase‐dUTP nick end labeling–positive nuclei: WT MI, 0.7±0.04%; ucp3−/− MI, 1.1±0.09%, P<0.05). Conclusions Our data indicate that ucp3 levels regulate reactive oxygen species levels and cell survival during hypoxia, modulating infarct size in the ischemic heart.

Neuropeptides and 5-HT immunoreactivity in the gastric nerves of the dogfish (Scyliorhinus stellaris)

The gastric autonomic innervation of the dogfish was examined for regulatory peptides and serotonin by immunochemical techniques. Bouins-fixed, paraffin-embedded or benzoquinone-fixed frozen sections were used for light microscopical immunocytochemistry and glutaraldehyde-fixed resin-embedded sections for electron microscopical immunocytochemistry. Bombesin-, somatostatin-, gastrin/cholecystokinin-, substance P-, peptide histidine isoleucine-, vasoactive intestinal peptide- and serotonin-immunoreactive nerves were found in all layers of the stomach wall. Bombesin and vasoactive intestinal peptide-containing nerves were identified at ultrastructural level. Radioimmunoassay of acetic acid extracts of tissue confirmed the presence of immunoreactivity for bombesin, somatostatin, substance P, peptide histidine isoleucine and vasoactive intestinal peptide. Reverse phase high performance liquid chromatography indicated that the peptides identified were broadly similar to their mammalian counterparts.

Co-localisation of substance P-, bombesin-and peptide histidine isoleucine (PHI)-like peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris

SummaryThe gastrointestinal tract of cartilaginous fishes, like that of higher vertebrates, is known to contain endocrine cells and nerves immunoreactive for a wide variety of peptides, some of which have been structurally characterised. Since we have found that substance P-, bombesin-and peptide histidine isoleucine-like immunoreactivities are similarly distributed in the endocrine cells of the dogfish pyloric stomach, we have tried to establish whether any of these peptides are co-localised. The cells were compared in thin serial sections with both ligh-and electron microscopical immunocytochemistry. Double immunolabelling was also used to show two immunoreactive peptides in the same tissue section. Further characterisation of the immunoreactivity was attempted by preabsorbing the antibodies with various peptides or synthetic fragments of peptide molecules. Immunoreactivity for all three peptides was frequently present in the same cells, whereas antibodies to other peptides such as gastrin and somatostatin marked different cells. Electron microscopy indicated that all the secretory granules in three morphologically different cell types reacted with antibodies to all three peptides. Dual localisation of unrelated peptides in endocrine cells or nerves is established in many cases, but triple localisation is as yet unusual. The immunoreaction for bombesin-like peptides is different in endocrine cells and nerves, indicating that dogfish bombesin may be present in two forms, in agreement with biochemical evidence.

Modulation of galanin and neuromedin U-like immunoreactivity in rat corticotropes after alteration of endocrine status.

The localization of galanin in rat lactotropes and human corticotropes is well established. Neuromedin U immunoreactivity is present in rat corticotropes but radioimmunoassay of thyroid-manipulated rat pituitaries has also linked it to the thyroid axis. We found galanin immunoreactivity in some rat corticotropes, so we have re-examined rat anterior pituitary galanin- and neuromedin U-like immunoreactivity by use of immunocytochemistry and electron microscopy in rats in the normal state and after estrogen administration or adrenalectomy. In normal rats galanin immunoreactivity was present in a few corticotropes and lactotropes, females showing more than males; neuromedin U-like immunoreactivity was present in some thyrotropes and most corticotropes, in both sexes. Where galanin, neuromedin U and ACTH immunoreactivities were colocalized in corticotropes they were present in the same granules. Estrogen administration caused an increase in number of galanin immunoreactive lactotropes, as previously shown. The proportion of neuromedin U-positive corticotropes was not affected. After adrenalectomy, only females showed a significant increase in the proportion of galanin-positive corticotropes. Neuromedin U immunoreactivity was significantly increased in both sexes, as previously shown. Thus, in rat, as in man, galanin can be present in corticotropes and its expression appears to be sexrelated. This finding, and the demonstration of thyrotrope neuromedin U (only examined in normal females), provide correlation with previous experiments. The influence of endocrine status on the expression of these novel peptides underlines the inherent plasticity of pituitary endocrine cells.