Laura Piqueras

Activation of PPARβ/δ Induces Endothelial Cell Proliferation and Angiogenesis

Objective—The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-&bgr;/&dgr; in endothelial cells remains unclear. Interestingly, the selective PPAR&bgr;/&dgr; ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPAR&bgr;/&dgr; in endothelial cell proliferation and angiogenesis. Methods and Results—Western blot analysis indicated PPAR&bgr;/&dgr; was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPAR&bgr;/&dgr; target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPAR&bgr;/&dgr;. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. Conclusions—PPAR&bgr;/&dgr; is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT1 and AT2 receptor-mediated P-selectin upregulation

BackgroundAngiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte–endothelial cell interactions in vivo. Methods and ResultsIntravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8±20.7 versus 16.4±3.1 cells/min), adhesion (11.4±1.0 versus 0.8±0.5 cells/100 &mgr;m), and emigration (4.0±0.7 versus 0.2±0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT1 (losartan) or AT2 (PD123,319) receptor antagonists significantly reduced Ang II–induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti–rat P-selectin monoclonal antibody abolished Ang II–induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation. ConclusionsAng II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

Trans- but Not Cis-Resveratrol Impairs Angiotensin-II–Mediated Vascular Inflammation through Inhibition of NF-κB Activation and Peroxisome Proliferator-Activated Receptor-γ Upregulation

Angiotensin II (Ang-II) displays inflammatory activity and is implicated in several cardiovascular disorders. This study evaluates the effect of cis- and trans (t)-resveratrol (RESV) in two in vivo models of vascular inflammation and identifies the cardioprotective mechanisms that underlie them. In vivo, Ang-II–induced arteriolar leukocyte adhesion was inhibited by 71% by t-RESV (2.1 mg/kg, i.v.), but was not affected by cis-RESV. Because estrogens influence the rennin-angiotensin system, chronic treatment with t-RESV (15 mg/kg/day, orally) inhibited ovariectomy-induced arteriolar leukocyte adhesion by 81%, partly through a reduction of cell adhesion molecule (CAM) expression and circulating levels of cytokine-induced neutrophil chemoattractant, MCP-1, and MIP-1α. In an in vitro flow chamber system, t-RESV (1–10 μM) undermined the adhesion of human leukocytes under physiological flow to Ang-II–activated human endothelial cells. These effects were accompanied by reductions in monocyte and endothelial CAM expression, chemokine release, phosphorylation of p38 MAPK, and phosphorylation of the p65 subunit of NF-κB. Interestingly, t-RESV increased the expression of peroxisome proliferator-activated receptor-γ in human endothelial and mononuclear cells. These results demonstrate for the first time that the in vivo anti-inflammatory activity of RESV is produced by its t-RESV, which possibly interferes with signaling pathways that cause the upregulation of CAMs and chemokine release. Upregulation of proliferator-activated receptor-γ also appears to be involved in the cardioprotective effects of t-RESV. In this way, chronic administration of t-RESV may reduce the systemic inflammatory response associated with the activation of the rennin-angiotensin system, thereby decreasing the risk of further cardiovascular disease.

Rolipram inhibits leukocyte-endothelial cell interactions in vivo through P- and E-selectin downregulation

Rolipram, a selective phosphodiesterase (PDE) type 4 inhibitor, was used to characterize leukocyte recruitment mechanisms in models of acute and subacute inflammation. Intravital microscopy within the rat mesenteric microcirculation was employed. Mesentery superfusion with PAF (0.1 μM) induced a significant increase in leukocyte rolling flux, adhesion and emigration at 60 min. Rolipram pretreatment, markedly inhibited these parameters by 100, 95 and 95% respectively. Similar effects were observed when the mesentery was superfused with LPS (1 μg ml−1) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment. LPS exposure of the mesentery for 4 h caused a greater increase in leukocyte rolling flux, adhesion and emigration which were inhibited by rolipram administration by 51, 71 and 81% respectively. Immunohistochemistry revealed a significant increase in P‐selectin expression after 60 min superfusion with PAF which was attenuated by rolipram. LPS exposure of the mesentery for 4 h caused a significant increase in P‐ and E‐selectin, intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression. Rolipram pretreatment down‐regulated both P‐ and E‐selectin expression but had no effect on ICAM‐1 and VCAM‐1 expression. Significant increases in plasma cyclic AMP levels were detected at 4.5 h after rolipram administration. In conclusion, we have demonstrated that rolipram is a potent in vivo inhibitor of leukocyte‐endothelial cell interactions. The effects observed are mediated through endothelial P‐ and E‐selectin downregulation. Therefore, selective PDE‐4 inhibitors may be useful in the control of different inflammatory disorders.

CXCR2 Blockade Impairs Angiotensin II–Induced CC Chemokine Synthesis and Mononuclear Leukocyte Infiltration

Objective—Angiotensin II (Ang-II) and mononuclear leukocytes are involved in atherosclerosis. This study reports the inhibition of Ang-II–induced mononuclear cell recruitment by CXCR2 antagonism and the mechanisms involved. Methods and Results—Ang-II (1 nmol/L, i.p. in rats) induced CXC and CC chemokines, followed by neutrophil and mononuclear cell recruitment. Administration of the CXCR2 antagonist, SB-517785-M, inhibited the infiltration of both neutrophils (98%) and mononuclear cells (60%). SB-517785-M had no effect on the increase in CXC chemokine levels but reduced MCP-1, RANTES, and MIP-1&agr; release by 66%, 63%, and 80%, respectively. Intravital microscopy showed that pretreatment with SB-517785-M inhibited Ang-II–induced arteriolar mononuclear leukocyte adhesion. Stimulation of human umbilical arterial endothelial cells (HUAECs) or whole blood with 1 &mgr;mol/L Ang-II induced the synthesis of chemokines. Ang-II increased HUAEC CXCR2 expression, and its blockade caused a significant reduction of MCP-1, -3, and RANTES release, as well as mononuclear cell arrest. Ang-II–induced MIP-1&agr; release from blood cells was also inhibited. Conclusion—Mononuclear leukocyte recruitment induced by Ang-II is, surprisingly, largely mediated by the CXC chemokines which appear to induce the release of CC chemokines. Therefore, CXC chemokine receptor antagonists may help to prevent mononuclear cell infiltration and the progression of the atherogenic process.

Somatostatin Receptor type 2 Mediates Bombesin-Induced Inhibition of Gastric Acid Secretion in Mice

Studies in isolated mouse stomach showed that bombesin releases somatostatin. We characterized the effects of exogenous bombesin on gastric acid secretion in mice and determined the involvement of somatostatin and somatostatin receptor type 2 (SSTR2) by using somatostatin immunoneutralization, the SSTR2 antagonist, PRL‐2903, and SSTR2 knockout mice. Gastric acid secretion was monitored under basal and pentagastrin‐, histamine‐ or bethanechol‐stimulated conditions in urethane‐anaesthetized mice. Bombesin (10–40 μg kg−1 h−1) and somatostatin‐14 (20 μg kg−1 h−1) were infused i.v. 10 and 30 min after PRL‐2903 or somatostatin antibody pretreatment, respectively. Urethane‐anaesthetized wild‐type mice had low basal acid secretion (0.12 ± 0.01 μmol (10 min)−1) compared with SSTR2 knockout mice (1.43 ± 0.10 μmol (10 min)−1). Somatostatin antibody and PRL‐2903 increased basal secretion in wild‐type mice but not in SSTR2 knockout animals. In wild‐type mice, bombesin inhibited secretagogue‐stimulated acid secretion in a dose‐dependent manner, and somatostatin‐14 inhibited pentagastrin‐stimulated secretion. In wild‐type mice pretreated with somatostatin antibody or PRL‐2903 and in SSTR2 knockout mice, bombesin and somatostatin‐14 i.v. infusion did not alter the increased gastric acid secretion. These results indicate that, in mice, bombesin inhibits gastric acid secretion through the release of somatostatin and the activation of SSTR2. These observations strengthen the important role of SSTR2 in mediating somatostatin inhibitory actions on gastric acid secretion.

Insulin resistance aggravates atherosclerosis by reducing vascular smooth muscle cell survival and increasing CX3CL1/CX3CR1 axis

AIMS Insulin resistance (IR) is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events are mainly due to rupture of unstable plaques, and the role of vascular smooth muscle cells (VSMCs) in this process in IR, Type 2 diabetes mellitus, and metabolic syndrome (T2DM/MetS) has not been fully addressed. Therefore, the role of VSMC survival in the generation of unstable plaques in T2DM/MetS and the involvement of inflammatory mediators was investigated. METHODS AND RESULTS Defective insulin receptor substrate 2 (IRS2)-mediated signalling produced insulin-resistant VSMCs with reduced survival, migration, and higher apoptosis than control cells. Silencing of IRS2 or inhibition of the V-akt murine thymomaviral oncogene homologue kinase (AKT)-extracellular signal-regulated kinase (ERK)-dependent pathway in VSMCs augmented expression of the inflammatory chemokine fractalkine (CX3CL1) and its receptor CX3CR1, previously involved in atheroma plaque vulnerability. Interestingly, treatment of VSMCs with CX3CL1 promoted apoptosis in the presence of other stimuli or when the AKT pathway was blocked. Analysis of a mouse model of IR-MetS and accelerated atherosclerosis, apoE-/-Irs2+/- mice, showed reduced VSMC survival, unstable plaques, and up-regulation of CX3CL1/CX3CR1 axis compared with apoE-/- mice. Human studies showed augmented soluble CX3CL1 plasma levels and CX3CR1 expression in monocytes from IR-MetS subjects compared with controls. A positive correlation between insulin levels, homeostatic model assessment (HOMA) index, carotid atherosclerosis, and CX3CR1 mRNA levels was also found in all patients. CONCLUSION IR increases plaque vulnerability by augmenting the CX3CL1/CX3CR1 axis, which is mechanistically linked to reduced VSMC survival. Thus, modulation of IRS2-dependent signalling emerges as a potential therapeutic strategy to promote VSMC survival and atheroma plaque stability and to reduce inflammatory mediators in IR-MetS.

2,3,9- and 2,3,11-Trisubstituted tetrahydroprotoberberines as D2 dopaminergic ligands

Dopamine-mediated neurotransmission plays an important role in relevant psychiatric and neurological disorders. Nowadays, there is an enormous interest in the development of new dopamine receptors (DR) acting drugs as potential new targets for the treatment of schizophrenia or Parkinsons disease. Previous studies have revealed that isoquinoline compounds such as tetrahydroisoquinolines (THIQs) and tetrahydroprotoberberines (THPBs) can behave as selective D2 dopaminergic alkaloids since they share structural similarities with dopamine. In the present study we have synthesized eleven 2,3,9- and 2,3,11-trisubstituted THPB compounds (six of them are described for the first time) and evaluated their potential dopaminergic activity. Binding studies on rat striatal membranes were used to evaluate their affinity and selectivity towards D1 and D2 DR and establish the structure-activity relationship (SAR) as dopaminergic agents. In general, all the tested THPBs with protected phenolic hydroxyls showed a lower affinity for D1 and D2 DR than their corresponding homologues with free hydroxyl groups. In previous studies in which dopaminergic affinity of 1-benzyl-THIQs (BTHIQs) was evaluated, the presence of a Cl into the A-ring resulted in increased affinity and selectivity towards D2 DR. This is in contrast with the current study since the existence of a chlorine atom into the A-ring of the THPBs caused increased affinity for D1 DR but dramatically reduced the selectivity for D2 DR. An OH group in position 9 of the THPB (9f) resulted in a higher affinity for DR than its homologue with an OH group in position 11 (9e) (250 fold for D2 DR). None of the compounds showed any cytotoxicity in freshly isolated human neutrophils. A molecular modelling study of three representative THPBs was carried out. The combination of MD simulations with DFT calculations provided a clear picture of the ligand binding interactions from a structural and energetic point of view. Therefore, it is likely that compound 9d (2,3,9-trihydroxy-THPB) behave as D2 DR agonist since serine residues cluster are crucial for agonist binding and receptor activation.

Retinoid X Receptor Agonists Impair Arterial Mononuclear Cell Recruitment through Peroxisome Proliferator-Activated Receptor-γ Activation

Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α–induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR agonists such as bexarotene or 9-cis-retinoid acid. RXR agonists also prevented TNF-α–induced VCAM-1 and ICAM-1 expression, as well as endothelial growth-related oncogene-α and MCP-1 release. Suppression of RXRα expression with a small interfering RNA abrogated these responses. Furthermore, inhibition of MAPKs and NF-κB pathways were involved in these events. RXR agonist-induced antileukocyte adhesive effects seemed to be mediated via RXRα/peroxisome proliferator-activated receptor (PPAR)γ interaction, since endothelial PPARγ silencing abolished their inhibitory responses. Furthermore, RXR agonists increased RXR/PPARγ interaction, and combinations of suboptimal concentrations of both nuclear receptor ligands inhibited TNF-α–induced mononuclear leukocyte arrest by 60–65%. In vivo, bexarotene dose-dependently inhibited TNF-α–induced leukocyte adhesion to the murine cremasteric arterioles and decreased VCAM-1 and ICAM-1 expression. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade. Thus, RXR agonists may constitute a new therapeutic tool in the control of the inflammatory process associated with cardiovascular disease.

Peripheral PACAP inhibits gastric acid secretion through somatostatin release in mice

Studies in rats suggest that PACAP modulates gastric acid secretion through the release of both histamine and somatostatin. We characterized the effects of exogenous PACAP on gastric acid secretion in urethane‐anesthetized mice implanted with a gastric cannula and in conscious 2‐h pylorus ligated mice, and determined the involvement of somatostatin and somatostatin receptor type 2 (SSTR2) by using somatostatin immunoneutralization, the SSTR2 antagonist, PRL‐2903, and SSTR2 knockout mice. Urethane‐anesthetized wild‐type mice had low basal acid secretion (0.10±0.01 μmol (10 min)−1) compared with SSTR2 knockout mice (0.93±0.07 μmol (10 min)−1). Somatostatin antibody and PRL‐2903 increased basal secretion in wild‐type mice but not in SSTR2 knockout animals. In wild‐type urethane‐anesthetized mice, PACAP‐38 (3–270 μg kg−1 h−1) did not affect the low basal acid secretion, but inhibited the acid response to pentagastrin, histamine, and bethanechol. In wild‐type urethane‐anesthetized mice pretreated with somatostatin antibody or PRL‐2903 and in SSTR2 knockout mice, peripheral infusion of PACAP‐38 or somatostatin‐14 did not inhibit the increased basal gastric acid secretion. In conscious wild‐type mice, but not in SSTR2 knockout mice, PACAP‐38 inhibited gastric acid secretion induced by 2‐h pylorus ligation. The antisecretory effect of PACAP‐38 was prevented by immunoneutralization of somatostatin. These results indicate that, in mice, peripheral PACAP inhibits gastric acid secretion through the release of somatostatin and the activation of SSTR2 receptors. There is no evidence for stimulatory effects of PACAP on acid secretion in mice.