Inmaculada Noguera

Menopause and Ovariectomy Cause a Low Grade of Systemic Inflammation that May Be Prevented by Chronic Treatment with Low Doses of Estrogen or Losartan

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-β-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1α and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1α in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-β-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis

BACKGROUND Implantation of a retrogradely-shed endometrium during menstruation requires an adequate blood supply. The endometrium has angiogenic potential, and endometriotic lesions grow in areas with a rich vascularization, suggesting that angiogenesis is a prerequisite for endometriosis development. Targeting vascular endothelial growth factor (VEGF) leads to an inhibition of endometriosis. Dopamine and its agonists, such as cabergoline (Cb2), promote VEGF receptor-2 (VEGFR-2) endocytosis in endothelial cells, preventing VEGF-VEGFR-2 binding and reducing neoangiogenesis. The aim of this study was to evaluate the anti-angiogenic properties of Cb2 on growth of established endometriosis lesions and investigate the molecular mechanisms by which Cb2 exerts the anti-angiogenic effect. METHODS Human endometrium fragments were implanted in female nude mice peritoneum, and mice were treated with vehicle, 0.05 or 0.1 mg/kg/day oral Cb2 for 14 days. After treatment, the implants were processed to assess proliferative activity, neoangiogenesis, VEGFR-2 phosphorylation and angiogenic gene expression. RESULTS A significant decrease in the percentage of active endometriotic lesions (P < 0.05) and cellular proliferation index (P < 0.001) was found with Cb2 treatment. Neoangiogenesis was reduced by Cb2 treatment, as observed at gross morphological level and by significant changes in gene expression. The degree of VEGFR-2 phosphorylation was significantly lower in Cb2-treated animals than controls. CONCLUSIONS Cb2 treatment in experimental endometriosis has an anti-angiogenic effect acting through VEGFR-2 activation. These findings support the testing of dopamine agonists as a novel therapeutic approach to peritoneal endometriosis in humans.

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1 The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves. 2 Vasopressin (10−10–10−7 M) caused concentration‐dependent contractions (pD2=8.36±0.09). The V1‐receptor antagonist d(CH2)5Tyr(Me)AVP (10−9–10−8 M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA2 value of 9.83 with a slope of 1.10±0.14. 3 Vasopressin (3×10 −10 and 10−9 M) caused concentration‐dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration‐response curve for noradrenaline. The V1‐receptor antagonist induced concentration‐dependent inhibitions of potentiation induced by vasopressin. The selective V1‐receptor agonist [Phe*, Orn8]‐vasotocin (3×10 −10 and 10−9 M) induced potentiation of electrical stimulation‐evoked responses which was also inhibited in the presence of the V1 antagonist (10−8 M). In contrast, the V2‐receptor agonist deamino‐8‐D‐arginine vasopressin (desmopressin 10−8–10−7 M) did not modify the electrical stimulation‐induced responses and the V2‐receptor antagonist [d(CH2)5, D‐Ile*, Ile4, Arg8]‐vasopressin (10−8–10−7 M) did not affect the potentiation evoked by vasopressin. 4 In artery rings contracted by 10−6 M noradrenaline in the presence of 10−6 M guanethidine and 10−6 M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency‐dependent relaxations which were unaffected by 10−9 M vasopressin but abolished by 10−6 M tetrodotoxin. 5 Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl2 to KCl depolarized vessels. The augmenting effects were inhibited by the V1 antagonist. 6 In the presence of the calcium antagonist nifedipine (10−6 M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl. 7 The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V1 receptor stimulation which brings about an increase in calcium entry through dihydropyridine‐sensitive calcium channels.

Right ventricular involvement in anterior myocardial infarction: a translational approach

AIMS The aim of the present study was to evaluate the involvement of the right ventricle (RV) in reperfused anterior ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS Left anterior descending (LAD)-perfused area (using thioflavin-S staining after selective infusion in proximal LAD artery, %), infarct size (using triphenyltetrazolium chloride staining, %), and salvaged myocardium (% of LAD-perfused area) in the right and left ventricle (LV) were quantified in a 90-min LAD occlusion 3-day reperfusion model in swine (n = 8). Additionally, we studied, using cardiovascular magnetic resonance, 20 patients with a first STEMI due to proximal LAD occlusion treated with primary angioplasty. Area at risk (T2-weighted sequence, %), infarct size (late enhancement imaging, %), and salvaged myocardium (% of area at risk) in the right and LV were quantified. In swine, a large LAD-perfused area was detected both in the right and LV (30 +/- 5 vs. 62 +/- 15%, P< 0.001) but more salvaged myocardium (94 +/- 6 vs. 73 +/- 11%, P< 0.001) resulted in a smaller right ventricular infarct size (2 +/- 1 vs. 16 +/- 5%, P< 0.001). Similarly, in patients a large area at risk was detected both in the right and LV (34 +/- 13 vs. 43 +/- 12%, P = 0.02). More salvaged myocardium (94 +/- 10 vs. 33 +/- 26%, P < 0.001) resulted in a smaller infarct size (2 +/- 3 vs. 30 +/- 16%, P< 0.001) in the RV. CONCLUSION In reperfused extensive anterior STEMI, a large area of the RV is at risk but the resultant infarct size is small.

Effect of ischemic postconditioning on microvascular obstruction in reperfused myocardial infarction. Results of a randomized study in patients and of an experimental model in swine.

BACKGROUND Ischemic postconditioning (PCON) appears as a potentially beneficial tool in ST-segment elevation myocardial infarction (STEMI). We evaluated the effect of PCON on microvascular obstruction (MVO) in STEMI patients and in an experimental swine model. METHODS A prospective randomized study in patients and an experimental study in swine were carried out in two university hospitals in Spain. 101 consecutive STEMI patients were randomized to undergo primary angioplasty followed by PCON or primary angioplasty alone (non-PCON). Using late gadolinium enhancement cardiovascular magnetic resonance, infarct size and MVO were quantified (% of left ventricular mass). In swine, using an angioplasty balloon-induced anterior STEMI model, MVO was defined as the % of area at risk without thioflavin-S staining. RESULTS In patients, PCON (n=49) in comparison with non-PCON (n=52) did not significantly reduce MVO (0 [0-1.02]% vs. 0 [0-2.1]% p=0.2) or IS (18 ± 13% vs. 21 ± 14%, p=0.2). MVO (>1 segment in the 17-segment model) occurred in 12/49 (25%) PCON and in 18/52 (35%) non-PCON patients, p=0.3. No significant differences were observed between PCON and non-PCON patients in left ventricular volumes, ejection fraction or the extent of hemorrhage. In the swine model, MVO occurred in 4/6 (67%) PCON and in 4/6 (67%) non-PCON pigs, p=0.9. The extent of MVO (10 ± 7% vs. 10 ± 8%, p=0.9) and infarct size (23 ± 14% vs. 24 ± 10%, p=0.8) was not reduced in PCON compared with non-PCON pigs. CONCLUSIONS Ischemic postconditioning does not significantly reduce microvascular obstruction in ST-segment elevation myocardial infarction. Clinical Trial Registration http://www.clinicaltrials.gov. Unique identifier: NCT01898546.

Estrogen receptor agonists and immune system in ovariectomized mice.

Several data implicate the immune system in bone lost after estrogen deficiency, however, some of the effects on the immune system of estrogen deficiency or of estrogen receptor (ER) modulation are not well established. In this study, the effect of ER agonists on the immune system in ovariectomized mice is analyzed. Mice were ovariectomized and were administered 17β-estradiol (E2), raloxifene (RAL) or genistein (GEN). The effect of a 4-week treatment on bone turnover and on several parameters that reflect the status of the immune system was studied. Results show that ovariectomy provoked both uterine atrophy and thymic hypertrophy. Although RAL corrected thymic hypertrophy, only E2 corrected both. Ovariectomized mice showed increased levels of serum calcium and cathepsin K gene expression and decreased levels of serum alkaline phosphatase (ALP) activity, which suggests that there is a persistent alteration in bone metabolism. Moreover, ovariectomy increased B-cells and CD25+ cells, and decreased the percentages of T-cells and Cbfa1 gene expression in bone marrow (BM). All ER agonists corrected, although to different degrees, changes induced by the ovariectomy. Furthermore, results showed that it is essential to adjust ER agonist doses to avoid immunosuppression, since all ER agonists decreased BM T-cell levels.

Endothelin-1-induced potentiation of adrenergic responses in the rabbit pulmonary artery: role of thromboxane A2

Abstract To examine whether low concentrations of endothelin-1 potentiate the vasocontrictor response to adrenergic stimulation, we recorded the isometric response of rings of rabbit pulmonary artery to electrical stimulation and noradrenaline. Endothelin-1 (10 −10 M) potentiated the contractions induced by electrical stimulation and noradrenaline. The endothelin ET B receptor antagonist (2,6-dimethylpiperidinecarbonyl-γ-methyl-Leu- N in -[Methoxycarbonyl]- d -Trp- d -Nle) (BQ-788, 10 −6 M), but not the endothelin ET A receptor antagonist cyclo( d -Asp-Pro- d -Val-Leu- d -TRP) (BQ-123, 10 −6 M), inhibited the potentiating effects of endothelin-1. Pretreatment with the cyclooxygenase inhibitor indomethacin, the thromboxane synthase inhibitor furegrelate and the thromboxane receptor antagonist [1 S -[1α,2α( Z ),3α,4α]]-7-[3-[[[[(1-oxoheptyl)amino]acetyl]amino] methyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid (SQ-30741) (all at 10 −5 M) prevented the potentiation induced by endothelin-1 on adrenergic stimulation. The Ca 2+ channel antagonist nifedipine (10 −6 M) did not affect the potentiation induced by endothelin-1. The results indicate that endothelin-1 potentiates the responses to electrical stimulation and noradrenaline by activating endothelin ET B receptors. This potentiation depends on the production of cyclooxygenase-generated factors, probably thromboxane A 2 .

Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization

Hydrodynamic injection has demonstrated to be very efficient in the liver of small animals, although this procedure must be translated to the clinical practice in a milder but no less efficient way. The present study evaluates the capacity of non‐invasive interventional catheterization as a procedure for naked DNA delivery to the heart in large animals.

Low RNA translation activit limits the efficacy of hydrodynamic gene transfer to pig liver in vivo

Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo.

Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

Introduction Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. Material and Methods A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. Results Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases. Conclusion Hydrofection of hAAT DNA to “in vivo” isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves.