Salvatore Salomone

Early release of HMGB-1 from neurons after the onset of brain ischemia

The nuclear protein high-mobility group box 1 (HMGB-1) promotes inflammation in sepsis, but little is known about its role in brain ischemia-induced inflammation. We report that HMGB-1 and its receptors, receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4, were expressed in normal brain and in cultured neurons, endothelia, and glial cells. During middle cerebral artery occlusion (MCAO), in mice, HMGB-1 immunostaining rapidly disappeared from all cells within the striatal ischemic core from 1 h after onset of occlusion. High-mobility group box 1 translocation from nucleus to cytoplasm was observed within the cortical periinfarct regions 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 predominantly translocated to the cytoplasm or disappeared in cells that colabeled with the neuronal marker NeuN. Furthermore, RAGE was robustly expressed in the periinfarct region after MCAO. Cellular release of HMGB-1 was detected by immunoblotting of cerebrospinal fluid as early as 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 released from neurons, in vitro, after glutamate excitotoxicity, maintained biologic activity and induced glial expression of tumor necrosis factor α (TNFα). Anti-HMGB-1 antibody suppressed TNFα upregulation in astrocytes exposed to conditioned media from glutamate-treated neurons. Moreover, TNFα and the cytokine intercellular adhesion molecule-1 increased in cultured glia and endothelial cells, respectively, after adding recombinant HMGB-1. In conclusion, HMGB-1 is released early after ischemic injury from neurons and may contribute to the initial stages of the inflammatory response.

Isoflavones: estrogenic activity, biological effect and bioavailability.

Isoflavones are phytoestrogens with potent estrogenic activity; genistein, daidzein and glycitein are the most active isoflavones found in soy beans. Phytoestrogens have similarity in structure with the human female hormone 17-β-estradiol, which can bind to both alpha and beta estrogen receptors, and mimic the action of estrogens on target organs, thereby exerting many health benefits when used in some hormone-dependent diseases. Numerous clinical studies claim benefits of genistein and daidzein in chemoprevention of breast and prostate cancer, cardiovascular disease and osteoporosis as well as in relieving postmenopausal symptoms. The ability of isoflavones to prevent cancer and other chronic diseases largely depends on pharmacokinetic properties of these compounds, in particular absorption and distribution to the target tissue. The chemical form in which isoflavones occur is important because it influences their bioavailability and, therefore, their biological activity. Glucose-conjugated isoflavones are highly polar, water-soluble compounds. They are hardly absorbed by the intestinal epithelium and have weaker biological activities than the corresponding aglycone. Different microbial families of colon can transform glycosylated isoflavones into aglycones. Clinical studies show important differences between the aglycone and conjugated forms of genistein and daidzein. The evaluation of isoflavone metabolism and bioavailability is crucial to understanding their biological effects. Lipid-based formulations such as drug incorporation into oils, emulsions and self-microemulsifying formulations have been introduced to increase bioavailability. Complexation with cyclodextrin also represent a valid method to improve the physicochemical characteristics of these substances in order to be absorbed and distributed to target tissues. We review and discuss pharmacokinetic issues that critically influence the biological activity of isoflavones.

Pronounced hypoperfusion during spreading depression in mouse cortex.

We studied unique cerebral blood flow (CBF) responses to cortical spreading depression in mice using a novel two-dimensional CBF imaging technique, laser speckle flowmetry. Cortical spreading depression caused a triphasic CBF response in both rat and mouse cortex. In rats, mild initial hypoperfusion (approximately 75% of baseline) was followed by a transient hyperemia reaching approximately 220% of baseline. In mice, the initial hypoperfusion was pronounced (40–50% of baseline), and the anticipated hyperemic phase barely reached baseline. The duration of hypoperfusion significantly correlated with the duration of the DC shift. As a possible explanation for the pronounced hypoperfusion, mouse cerebral vessels showed enhanced resistance to relaxation by acetylcholine (3 μM) after K+-induced preconstriction (20, 40, and 80 mM) but dilated normally in response to acetylcholine after preconstriction with U46619, a synthetic thromboxane A2 analog. By contrast, rat vessels dilated readily to acetylcholine after preconstriction by K+. The transient normalization of CBF after hypoperfusion in the mouse was abolished by L-NA but not 7-NI. In summary, the CBF response to cortical spreading depression in mice contrasts with the rat in that the initial hypoperfusion is pronounced, and the hyperemic phase is markedly diminished. The differences in CBF response between species may be in part caused by an increased sensitivity of mouse cerebral vessels to elevated extracellular K+.

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.

S1P3 receptors mediate the potent constriction of cerebral arteries by sphingosine-1-phosphate.

We characterized the effect of Sphingosine-1-phosphate (S1P) on vascular tone. S1P selectively constricted isolated cerebral, but not peripheral arteries, despite ubiquitous expression of S1P(1), S1P(2), S1P(3) and S1P(5) receptor mRNA. Clostridium B and C3 toxins and the rho-kinase inhibitor Y27632 (trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexane carboxamide) reduced this vasoconstriction to S1P, indicating that the response was mediated through Rho. Pertussis toxin displayed only weak inhibition, suggesting minor involvement of G(i/o) protein. The S1P effect was specifically reduced by adenovirus bearing a s1p(3) but not s1p(2), antisense construct. Furthermore, suramin, which selectively blocks S1P(3) receptors, inhibited the vasoconstrictor effect of S1P, indicating that S1P(3) receptors account for at least part of S1P-mediated vasoconstriction in cerebral arteries. In vivo, intracarotid injection of S1P decreased cerebral blood flow, an effect prevented by suramin treatment. Because S1P constricts cerebral blood vessels and is released from platelets during clotting, the S1P/S1P(3) system constitutes a novel potential target for cerebrovascular disease therapy.

Analysis of sphingosine 1-phosphate receptors involved in constriction of isolated cerebral arteries with receptor null mice and pharmacological tools

Sphingosine 1‐phosphate (S1P) selectively and potently constricts isolated cerebral arteries, but this response has not been pharmacologically characterized.

Obesity increases vascular senescence and susceptibility to ischemic injury through chronic activation of Akt and mTOR.

Chronic activation of Akt and mammalian target of rapamycin (mTOR) links diet-induced obesity with cardiovascular disease. Akting on the Vasculature Although obesity is a well-known risk factor for cardiovascular disease, the signals that connect the two remain unclear. Noting that aberrant signaling involving mammalian target of rapamycin (mTOR) and Akt has been linked to obesity and its pathophysiological complications and that both of these kinases have been implicated in development of cellular senescence, Wang et al. explored the roles of Akt and mTOR in endothelial cell senescence. They showed that increased endothelial Akt signaling linked a high-fat diet to increased endothelial cell senescence and vascular dysfunction in mice. Intriguingly, the mTOR inhibitor rapamycin inhibited the long-term activation of endothelial Akt, as well as vascular cell senescence, and ameliorated the vascular sequelae of ischemia. The authors thus propose that inhibition of Akt activation with rapamycin therapy may have clinical benefits in obesity-related cardiovascular disease. Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR)–rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease.

Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS−/–) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS−/– mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS−/– mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Regulation of Endothelial Nitric Oxide Synthase and Postnatal Angiogenesis by Rac1

Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1+/−) using Cre-loxP technology. The EC-Rac1+/− mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1+/flox) mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1+/− mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of l-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction.

Selectivity scale of calcium antagonists in the human cardiovascular system based on in vitro studies

The inhibitory effect of calcium antagonists has been studied on the calcium signal and on contractile activity in the human coronary and internal mammary arteries. We observed that rhythmic vasospasms, either spontaneous or evoked by serotonin and endothelin, were highly sensitive to calcium-channel inhibitors. Functional parameters describing the inhibition have been compared to binding parameters estimated in radioligand studies on membranes prepared from human coronary artery and from human heart. Taking the present studies and observations already published together, it was possible to build up a selectivity scale for diltiazem, verapamil, nifedipine, and nisoldipine. This showed that nisoldipine was more selective for coronary artery than the other calcium antagonists so far studied. Such a selectivity observed in functional studies on intact human preparations in vitro could be predicted considering the kinetic parameters of the interaction of nisoldipine with calcium channels.