Andrea Cignarella

Gender Differences in Endothelial Progenitor Cells and Cardiovascular Risk Profile The Role of Female Estrogens

Objective—Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state. Methods and Results—As measured by flow-cytometry in 210 healthy subjects, CD34+KDR+ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17&bgr;-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo. Conclusions—EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.

Phenotype-dependent differences in apolipoprotein E metabolism and in cholesterol homeostasis in human monocyte-derived macrophages.

In this study, we investigated the impact of the common apoE polymorphism on apoE metabolism and cholesterol homeostasis in monocyte-derived macrophages isolated from E2/2, E3/3, and E4/4 subjects. Unloaded cells of all genotypes contained similar amounts of free cholesterol, cholesteryl ester, and apoE mRNA. E3/3 cells secreted 77 and 30% more apoE than E2/2 or E4/4 cells, respectively. Pulse-chase studies confirmed that the apoE secretion rate was greatest in E3/3 and least in E2/2 cells and showed that a portion of apoE2, but not apoE3 or apoE4, was degraded intracellularly. Surface binding of apoE was greatest in E4/4 cells, as revealed by heparinase treatment. On cholesterol loading with acetylated LDL, apoE mRNA levels and protein secretion rose most in E4/4 and least in E2/2 cells. Cholesterol and cholesteryl ester content, however, rose most in E2/2 and least in E3/3 cells. Incubations with 3H-cholesterol-labeled acetylated LDL revealed that E2/2 cells were most efficient at secreting cholesterol. The greatest reuptake of 3H-cholesterol-rich particles was from E4/4 macrophage- conditioned media. Thus, E2/2 macrophages, despite a low apoE secretion rate, are protected from cholesterol storage by apoE-mediated cholesterol efflux. In E3/3 macrophages, cholesterol accumulation is lessened by a high basal apoE secretion rate. E4/4 macrophages secrete the most apoE but lack effective net cholesterol efflux due to enhanced surface binding and reuptake of cholesterol-rich particles.

ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages

Apolipoprotein E (apoE) produced by macrophages in the arterial wall protects against atherosclerosis, but the regulation of its secretion by these cells is poorly understood. Here we investigated the contribution of the adenosine triphosphate binding cassette transporters ABCA1 and ABC8 to the secretion of apoE from either primary human monocyte‐derived macrophages (HMDM) or human THP1 macrophages. During incubations of up to 6 h, apoE secretion from both THP1 macrophages and HMDM was stimulated by 8‐Br‐cAMP, which activates ABCA1 expression. The putative ABCA1 inhibitor glyburide and antisense oligonucleotides directed against ABCA1 mRNA significantly reduced apoE secretion from THP1 macro‐phages and HMDM. Antisense oligonucleotides directed against ABC8 mRNA also inhibited apoE secretion, although this inhibition was less pronounced and consistent than in the case of ABCA 1. ApoE secretion from HMDM of ABCA1‐deficient patients with Tangier disease was also decreased. ApoE mRNA expression was not affected by inhibition of ABCA1 or ABC8 in normal HMDM or the lack of functional ABCA1 in HMDM from Tangier disease patients. Inhibition of ABCA1 in HMDM prevented the occurrence of anti‐apoE‐immunoreactive granular structures in the plasma membrane. We conclude that ABCA1 and, to a lesser extent, ABC8 both promote secretion of apoE from human macrophages.—von Eckardstein, A., Langer, C., Engel, T., Schaukal, I., Cignarella, A., Reinhardt, J., Lorkowski, S., Li, Z., Zhou, X., Cullen, P., Assmann, G. ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte‐derived macrophages. FASEB J. 15, 1555–1561 (2001)

NOVEL LIPID-LOWERING PROPERTIES OF VACCINIUM MYRTILLUS L. LEAVES, A TRADITIONAL ANTIDIABETIC TREATMENT, IN SEVERAL MODELS OF RAT DYSLIPIDAEMIA: A COMPARISON WITH CIPROFIBRATE

Vaccinium myrtillus L. (blueberry) leaf infusions are traditionally used as a folk medicine treatment of diabetes. To further define this therapeutical action, a dried hydroalcoholic extract of the leaf was administered orally to streptozotocin-diabetic rats for 4 days. Plasma glucose levels were consistently found to drop by about 26% at two different stages of diabetes. Unexpectedly, plasma triglyceride (TG) were also decreased by 39% following treatment. Subsequent to the latter observation, possible lipid-lowering properties of the extract were investigated on other models of hyperlipidaemia and ciprofibrate, a well-established hypolipidaemic drug, was used as a reference compound. Both drug reduced TG levels of rats on hyperlipidaemic diet in a dose-dependent fashion. When administered at single doses over the same experimental period, blueberry and ciprofibrate were effective in lowering TG concentrations in ethanol-treated normolipidaemic animals and in genetically hyperlipidaemic Yoshida rats. Unlike ciprofibrate, however, blueberry failed to prevent the rise in plasma TG elicited by fructose and did not affect free fatty acid levels in any of the above experimental conditions. In rats treated with Triton WR-1339, blueberry feeding induced an hypolipidaemic activity one hour after injection but proved to be ineffective at later time points, thus suggesting that its hypolipidaemic action may reflect improved TG-rich lipoprotein catabolism. In addition, ciprofibrate and the extract were tested for antithrombotic activity using a collagen-triggered model of venous thrombosis in diabetic and Yoshida rats. Only ciprofibrate, however, significantly reduced thrombus formation in diabetics, possibly because of its effects on free fatty acid metabolism, whereas no effect was observed in Yoshida rats. In conclusion, the present findings indicate that active consituent(s) of Vaccinium myrtillus L. leaves may prove potentially useful for treatment of dyslipidaemiae associated with impaired TG-rich lipoprotein clearance.

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy

Aim/hypothesisMonocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation.MethodsWe enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68+CCR2+) and M2 (CX3CR1+CD206+/CD163+) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals.ResultsWe found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals.Conclusions/interpretationWe show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats.

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens.

Diabetes Undermines Estrogen Control of Inducible Nitric Oxide Synthase Function in Rat Aortic Smooth Muscle Cells Through Overexpression of Estrogen Receptor-β

Background—Previous reports from our group have shown that 17&bgr;-estradiol reduces the synthesis and activity of inducible nitric oxide synthase (iNOS) in rat aortic smooth muscle cells (SMC) in response to inflammatory mediators. In this study, we investigated the effect of 17&bgr;-estradiol on iNOS function in aortic SMC from streptozotocin-diabetic rats. Methods and Results—Comparative analysis of NO release and of iNOS mRNA and protein content after 24-hour stimulation with a cytokine mixture revealed milder iNOS activation in diabetic than in control SMC. Furthermore, 17&bgr;-estradiol dose-dependently blocked iNOS synthesis and activity in control but not in diabetic SMC. The defective estrogen response in diabetic SMC at 24 hours could not be attributed to reduced expression of estrogen receptors (ER). In fact, mRNA and protein levels of ER&agr; and, to a greater extent, of ER&bgr;, were increased in diabetic compared with nondiabetic SMC. Cytokines decreased ER&agr; and ER&bgr; expression in both groups. However, 17&bgr;-estradiol dose-dependently restored the expression of ER&agr; but further downregulated that of ER&bgr;, indicating a differential regulation of ER isoforms. Conclusions—Estrogenic control of iNOS was impaired in diabetic SMC. This was associated with a larger increase of ER&bgr; than of ER&agr; protein, whereas 17&bgr;-estradiol regulated the two isoforms in an opposite fashion. Thus, modifications in the estrogen modulation of iNOS and in the expression pattern of ER may be involved in diabetic vascular dysfunction.

Prolonged Ovarian Hormone Deprivation Impairs the Protective Vascular Actions of Estrogen Receptor αAgonists

The vascular consequences of estrogen treatment may be driven by its initiation timing. We tested the hypothesis that the duration of ovarian hormone deprivation before estrogen reintroduction affects the role of estrogen as mediator of endothelial function and vascular relaxation in nondiseased vessels. Rats were ovariectomized and implanted with 17&bgr;-estradiol (E2) or oil capsules 1, 4, and 8 months after surgery. After the longest hypoestrogenicity period, acetylcholine-mediated aortic relaxation was attenuated and insensitive to E2 administration despite endothelial integrity. Whereas no rapid vasorelaxant responses were elicited by an estrogen receptor (ER) &bgr;–selective agonist, responses to E2 and an ER&agr; selective agonist waned postovariectomy at any given time and were restored by E2 treatment after 1 and 4 months but not 8 months postovariectomy. Accordingly, endothelial ER&agr; mRNA and protein expression declined ≈6-fold after prolonged hypoestrogenicity and was restored by estrogen replacement starting 1 month but not 8 months postovariectomy. Furthermore, the amount of active phosphorylated endothelial NO synthase rose significantly after E2 replacement after 1 and 4 months but not 8 months postovariectomy. The present findings document that the functional impairment of the ER&agr;/endothelial NO synthase signaling network after an extended period of hypoestrogenicity was not restored by E2 administration, providing experimental support to early initiation of estrogen replacement with preferential ER&agr; targeting to improve cardiovascular outcomes.

Diabetes abolishes the vascular protective effects of estrogen in female rats

Estrogen is known to exert a protective effect against cardiovascular disease. However, women with diabetes have three times the risk as compared with age-matched non-diabetic women. Our previous study on aortic rings of ovariectomized (OVX) female rats treated with 17-beta-estradiol (E2) demonstrated that the beneficial effect of estrogen is related to the basal release of NO from endothelial cells. In the present study, in order to understand why estrogen protection is abolished in diabetes, we tested vascular responses in OVX, streptozotocin-diabetic female rats and their non-diabetic controls receiving or not E2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E2-treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E2-deprived rats. The basal release of NO, as evaluated by concentration-related responses to N(G)-methyl-L-arginine acetate in NE-precontracted aortic rings, was found to be impaired in E2-treated diabetic rats, no further effect being induced by E2 deprivation. The endothelium-dependent relaxation produced by carbachol did not change between groups, whereas the relaxation produced by histamine was enhanced by both diabetes and E2 deprivation. However, E2 treatment counteracted the response to histamine only in preparations from non-diabetic animals. Finally, the relaxation induced by sodium nitroprusside, an endothelium-independent relaxant agent, was comparable between groups. These findings suggest that the lack of protective effects of estrogen in diabetes may be mainly ascribed to the failure of estrogen to reverse the impaired basal release of NO and the abnormal relaxation to histamine, which are observed in the aorta of diabetic rats.

Selective Agonists of Estrogen Receptor Isoforms. New Perspectives for Cardiovascular Disease

The cloning of estrogen receptors (ERs) and generation of ER-deficient mice have increased our understanding of the molecular mechanisms underlying the cardiovascular effects of estrogen. It is conceivable that clinical trials of estrogens so far failed to improve cardiovascular health because of the poor ER isoform selectivity and tissue specificity of endogenous hormones as well as incorrect treatment timing and regimens. Tissue-selective ER modulators (SERMs) may be safer agents than endogenous estrogens for cardiovascular disease. Yet, designing isoform-selective ER ligands (I-SERMs) with agonist or antagonist activity is required to pursue improved pharmacological control of ERs, especially taking into account emerging evidence for the beneficial role of vascular ER&agr; activation. Ideally, the quest for unique ER ligands targeted to the vascular wall should lead to compounds that merge the pharmacological profiles of SERM and I-SERM agents. This review highlights the current bases for and approaches to selective ER modulation in the cardiovascular system.