Recent evidence suggests that besides its action on the central nervous system, leptin can modulate vascular tone through local mechanisms involving nitric oxide (NO) release. In this study, using a fluorescent probe for direct determination of NO, we demonstrated both in endothelial cells and in vessels that leptin is able to stimulate NO release. The effect of leptin on NO is abolished by erbstatin A, a Ca²⁺-independent tyrosine kinase inhibitor, whereas it is not influenced by calcium removal or by other protein phosphorylation inhibitors, such as genistein (an ATP-dependent tyrosine-kinase inhibitor) or wortmannin and LY294002 (two different phosphatidylinositol [PI] 3-kinase inhibitors). Accordingly, leptin-induced vasorelaxation in aortic rings was abolished only by erbstatin A. Furthermore, immunoblotting studies revealed that leptin evokes Akt phosphorylation, with a comparable time course in both endothelial cells and vessels. Also in this experimental system, the effect of leptin was abolished by erbstatin A and not by other inhibitors. Finally, a considerable increase in endothelial NO synthase (eNOS) phosphorylation in Ser¹¹⁷⁷ was found when vessels were treated with leptin. In conclusion, leptin induces NO production by activating a PI 3-kinase–independent Akt-eNOS phosphorylation pathway.

Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein–coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)γ are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kγ was found to play a role in angiotensin II–evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kγ was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kγ was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca²⁺ channel–mediated extracellular Ca²⁺ entry. These data indicate that PI3Kγ is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kγ function might be exploited to improve therapeutic intervention on hypertension.

Arterial hypertension is not only a major risk factor for cerebrovascular accidents, such as stroke and cerebral hemorrhage, but is also associated to milder forms of brain injury. One of the main causes of neurodegeneration is the increase in reactive oxygen species (ROS) that is also a common trait of hypertensive conditions, thus suggesting that such a mechanism could play a role even in the onset of hypertension-evoked brain injury. To investigate this issue, we have explored the effect of acute-induced hypertensive conditions on cerebral oxidative stress. To this aim, we have developed a mouse model of transverse aortic coarctation (TAC) between the two carotid arteries, which imposes acutely on the right brain hemisphere a dramatic increase in blood pressure. Our results show that hypertension acutely induced by aortic coarctation induces a breaking of the blood–brain barrier (BBB) and reactive astrocytosis through hyperperfusion, and evokes trigger factors of neurodegeneration such as oxidative stress and inflammation, similar to that observed in cerebral hypoperfusion. Moreover, the derived brain injury is mainly localized in selected brain areas controlling cognitive functions, such as the cortex and hippocampus, and could be a consequence of a defect in the BBB permeability. It is noteworthy to emphasize that, even if these latter events are not enough to produce ischemic/hemorrhagic injury, they are able to alter mechanisms fundamental for maintaining normal brain function, such as protein synthesis, which has a prominent role for memory formation and cortical plasticity.

Alzheimer's disease (AD) has been recently associated with vascular risk factors. β-amyloid peptides (AβP), the main component of senile plaques typical of AD, circulate in soluble globular form in bloodstream. Interestingly, AβP is able to induce endothelial dysfunction, and this effect may represent the link between vascular and neuronal pathophysiological factors involved in AD. We aimed to clarify the molecular mechanisms underlying globular AβP-induced vascular toxicity. Using several methodological approaches, we have observed that in vascular tissues globular AβP is unable to induce oxidative stress, one of the mechanisms hypothesized involved in β-amyloid toxicity. More important, we have demonstrated that globular AβP is able to localize on vascular endothelium, where it inhibits eNOS enzymatic activity. In particular, AβP enhances eNOS phosphorylation on threonine 495 and serine 116 and reduces acetylcholine-induced phosphorylation on serine 1177. Such an effect depends on a PKC signaling pathway, as suggested by its phosphorylation on serine 660. In fact, selective inhibition of the calcium-dependent group of PKC is able to rescue β-amyloid-induced alteration of eNOS phosphorylation, NO production, and endothelial vasorelaxation. The activation of these Ca²⁺-dependent pathways is probably due to the ability of AβP to evoke Ca²⁺ leakage from inositol 1,4,5-triphosphate receptors on endoplasmic reticulum. Our data demonstrate that globular AβP-induced endothelial NO dysfunction can be attributed to an alteration of intracellular Ca²⁺ homeostasis, which could lead to the activation of calcium-dependent group of PKC with a consequent change of the eNOS phosphorylation pattern. These mechanisms could contribute to shed further light on the toxic effect of β-amyloid in vascular tissues.

Hypertension can lead to subarachnoid hemorrhage and eventually to cerebral vasospasm. It has been suggested that the latter could be the result of oxidative stress and an inflammatory response evoked by subarachnoid hemorrhage. Because an unavoidable consequence of hemorrhage is lysis of red blood cells, we first tested the hypothesis on carotid arteries that the proinflammatory cytokine tumor necrosis factor-α contributes to vascular oxidative stress evoked by hemolysis. We observed that hemolysis induces a significant increase in tumor necrosis factor-α both in blood and in vascular tissues, where it provokes Rac-1/NADPH oxidase–mediated oxidative stress and vasoconstriction. Furthermore, we extended our observations to cerebral vessels, demonstrating that tumor necrosis factor-α triggered this mechanism on the basilar artery. Finally, in an in vivo model of subarachnoid hemorrhage obtained by the administration of hemolyzed blood in the cisterna magna, we demonstrated, by high-resolution ultrasound analysis, that tumor necrosis factor-α inhibition prevented and resolved acute cerebral vasoconstriction. Moreover, tumor necrosis factor-α inhibition rescued the hemolysis-induced brain injury, evaluated with the method of 2,3,5-triphenyltetrazolium chloride and by the histological analysis of pyknotic nuclei. In conclusion, our results demonstrate that tumor necrosis factor-α plays a crucial role in the onset of hemolysis-induced vascular injury and can be used as a novel target of the therapeutic strategy against cerebral vasospasm.