Gennaro Galasso

Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis

Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Akt1-/-) mice also have reduced endothelial progenitor cell (EPC) mobilization in response to ischemia, and reintroduction of WT EPCs, but not EPCs isolated from Akt1-/- mice, into WT mice improves limb blood flow after ischemia. Mechanistically, the loss of Akt1 reduces the basal phosphorylation of several Akt substrates, the migration of fibroblasts and ECs, and NO release. Reconstitution of Akt1-/- ECs with Akt1 rescues the defects in substrate phosphorylation, cell migration, and NO release. Thus, the Akt1 isoform exerts an essential role in blood flow control, cellular migration, and NO synthesis during postnatal angiogenesis.

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.

Impaired Angiogenesis in Glutathione Peroxidase-1–Deficient Mice Is Associated With Endothelial Progenitor Cell Dysfunction

Several vascular disease are characterized by elevated levels of reactive oxygen species (ROS). Vascular endothelium is protected from oxidant stress by expressing enzymes such as glutathione peroxidase type 1 (GPx-1). In this study, we investigated the effect of vascular oxidant stress on ischemia-induced neovascularization in a murine model of homozygous deficiency of GPx-1. GPx-1–deficient mice showed impaired revascularization following hindlimb ischemic surgery based on laser Doppler measurements of blood flow and capillary density in adductor muscle. GPx-1–deficient mice also showed an impaired ability to increase endothelial progenitor cell (EPC) levels in response to ischemic injury or subcutaneous administration of vascular endothelial growth factor protein. EPCs isolated from GPx-1–deficient mice showed a reduced ability to neutralize oxidative stress in vitro, which was associated with impaired migration toward vascular endothelial growth factor and increased sensitivity to ROS-induced apoptosis. EPCs isolated from GPx-1–deficient mice were impaired in their ability to promote angiogenesis in wild-type mice, whereas wild-type EPCs were effective in stimulating angiogenesis in GPx-1–deficient mice. These data suggest that EPC dysfunction is a mechanism by which elevated levels of ROS can contribute to vascular disease.

Impaired Revascularization in a Mouse Model of Type 2 Diabetes Is Associated With Dysregulation of a Complex Angiogenic-Regulatory Network

Objective—Diabetes is a risk factor for the development of cardiovascular diseases associated with impaired angiogenesis or increased endothelial cell apoptosis. Methods and Results—Here it is shown that angiogenic repair of ischemic hindlimbs was impaired in Leprdb/db mice, a leptin receptor–deficient model of diabetes, compared with wild-type (WT) C57BL/6 mice, as evaluated by laser Doppler flow and capillary density analyses. To identify molecular targets associated with this disease process, hindlimb cDNA expression profiles were created from adductor muscle of Leprdb/db and WT mice before and after hindlimb ischemia using Affymetrix GeneChip Mouse Expression Set microarrays. The expression patterns of numerous angiogenesis-related proteins were altered in Leprdb/db versus WT mice after ischemic injury. These transcripts included neuropilin-1, vascular endothelial growth factor-A, placental growth factor, elastin, and matrix metalloproteinases implicated in blood vessel growth and maintenance of vessel wall integrity. Conclusion—These data illustrate that impaired ischemia-induced neovascularization in type 2 diabetes is associated with the dysregulation of a complex angiogenesis-regulatory network.

Adiponectin promotes endothelial progenitor cell number and function

Obesity‐linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose‐derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN‐KO) in response to hindlimb ischemia. Adenovirus‐mediated delivery of adiponectin increased EPC levels in both WT and APN‐KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity‐related cardiovascular disorders.

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.

Role of β2 Adrenergic Receptors in Human Atherosclerotic Coronary Arteries

Background—Adrenergic regulation of coronary vasomotion is balanced between &agr;1-adrenergic–mediated (&agr;1-AR) constriction and &bgr;2-adrenergic–mediated (&bgr;2-AR) relaxation. This study aimed at assessing the role of &bgr;2-ARs in normal, mildly atherosclerotic, and stenotic human coronary arteries. Methods and Results—During intracoronary (IC) infusion of increasing doses of the &bgr;2-AR agonist salbutamol (0.15, 0.3, and 0.6 &mgr;g/min) and the endothelial vasodilator acetylcholine (1, 3, and 10 &mgr;g/min), we measured (1) changes in lumen diameter (LD) by quantitative coronary angiography in 34 normal, 55 mildly atherosclerotic, and 42 stenotic coronary artery segments and (2) changes in average peak velocity (APV) and coronary blood flow (CBF) with the use of Doppler flow wire in 11 normal, 10 mildly atherosclerotic, and 11 stenotic coronary arteries. In 6 of 11 stenotic coronary arteries, the protocol was repeated after an IC bolus (12 &mgr;g/kg) of the &agr;-adrenergic blocker phentolamine. In 6 of 11 normal coronary arteries, the protocol was repeated after an IC infusion (60 &mgr;mol/min) of NG-monomethyl-l-arginine (L-NMMA), a nitric oxide inhibitor. Neither salbutamol IC infusion nor acetylcholine significantly changed heart rate or blood pressure, whereas L-NMMA slightly increased blood pressure. In normal coronary arteries, salbutamol increased LD (LD max %: 11±2, P<0.05), APV (APV max %: 53±17, P<0.05), and CBF (CBF max %: 57±17, P<0.05), whereas L-NMMA caused a blunted APV (APV max %: 27±6, P<0.05) and CBF (CBF max %: 29±6, P<0.05) response to salbutamol. In mildly atherosclerotic coronary arteries, the salbutamol increase in LD (LD max %: 10±2, P<0.05), APV (APV max %: 33±12, P<0.05), and CBF (CBF max %: 37±12, P<0.05) was preserved. In stenotic coronary arteries, salbutamol induced a paradoxical reduction in LD (LD max %: −6±2, P<0.05), APV (APV max %: −15±9, P<0.05), and CBF (CBF max %: −15±6, P<0.05), which was no longer observed after phentolamine. Acetylcholine increased LD (LD max %: 14±3, P<0.05), APV (APV max %: 61±20, P<0.05), and CBF (CBF max %: 67±19, P<0.05) in normal coronary arteries. In mildly atherosclerotic coronary arteries, acetylcholine induced a significant reduction in LD (LD max %: −15±2, P<0.05) and no changes in APV (APV max %: −6±13, P=NS) and CBF (CBF max %: −10±13, P=NS). In stenotic coronary arteries, acetylcholine significantly reduced LD (LD max %: −15±3, P<0.05), APV (APV max %: −15±9, P<0.05), and CBF (CBF max %: −15±6, P<0.05). Conclusions—In severely atherosclerotic coronary arteries, &bgr;2-adrenergic vasodilatation is impaired, and this might contribute to alter the vasomotor balance, further precipitating myocardial ischemia during sympathetic activation.

Impact of Diabetes on Cardiac Sympathetic Innervation in Patients With Heart Failure: A 123I meta-iodobenzylguanidine (123I MIBG) scintigraphic study

OBJECTIVE Impaired parasympathetic and sympathetic nervous system activity have been demonstrated in patients with diabetes mellitus (DM) and correlated with worse prognosis. Few data are available on the effect of DM on cardiac neuropathy in heart failure (HF). The aim of the current study was to assess cardiac sympathetic activity in HF patients with and without DM. RESEARCH DESIGN AND METHODS Patients with severe HF (n = 75), with (n = 37) and without DM (n = 38), and 14 diabetic patients with normal cardiac function underwent 123I meta-iodobenzylguanidine scintigraphy from which early and late heart-to-mediastinum (H/M) ratios were calculated. Clinical, echocardiographic, and biochemical data were measured. RESULTS DM compared with non-DM patients showed significantly lower early (1.65 ± 0.21 vs. 1.75 ± 0.21; P < 0.05) and late H/M ratios (1.46 ± 0.22 vs. 1.58 ± 0.24; P < 0.03). Early and late H/M were significantly higher in DM patients without HF (2.22 ± 0.35 and 1.99 ± 0.24, respectively) than HF patients with (P < 0.0001) and without (P < 0.0001) DM. In HF patients, an inverse correlation between early or late H/M ratio and hemoglobin A1c (HbA1c) (Pearson = −0.473, P = 0.001; Pearson = −0.382, P = 0.001, respectively) was observed. In DM, in multivariate analysis, HbA1c and ejection fraction remained significant predictors of early H/M; HbA1c remained the only significant predictor of late H/M. No correlation between early or late H/M and HbA1c was found in non-DM patients. CONCLUSIONS Diabetic patients with HF show lower cardiac sympathetic activity than HF patients not having DM or than DM patients with a similar degree of autonomic dysfunction not having HF. HbA1c correlated with the degree of reduction in cardiac sympathetic activity.

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.