Ruoli distinti delle due isoforme di recettori degli estrogeni nella disfunzione vascolare in modelli animali di diabete

Recent large-scale clinical trials found a significantly lower incidence of diabetes in postmenopausal women on hormone replacement therapy despite no improvement in vascular outcomes. The mechanisms accounting for this outcome, however, are largely unknown although estrogen is increasingly recognized as an important regulator of glucose homeostasis. Because the endogenous hormone binds to its receptors with identical affinity, the metabolic effects of individual estrogen receptor (ER) isoforms, ERa and ERb, are hard to entangle and appear to be tissue- and species-specific. Accordingly, there is little information as to how ERa and ERb affect the course and timeline of diabetic vascular dysfunction, which ultimately results in macrovascular complications of clinical relevance. We previously demonstrated that estrogen’s anti-inflammatory activity is attenuated in vascular smooth muscle cells (SMCs) from streptozotocin (STZ)- diabetic rats, which display ERb overexpression with respect to normoglycemic controls. The biological significance of ERb overexpression in vascular cells from diabetic rats is as yet unclear. Data from our group indicate that moderately selective ERb agonists induce the expression of inflammatory enzymes in rat vascular SMCs. There is also evidence that ERa mediates protective antiinflammatory effects in vascular and nonvascular tissues including pancreatic b- cells. ERa-selective, but not ERb-selective agonists improve vascular function by mediating vessel relaxation at concentrations where receptor selectivity is maintained. Based on this knowledge, we set out to assess the relative contribution of ER isoforms to the progression of experimental insulin-deficient diabetes and the accompanying vascular dysfunction in rodents through a comprehensive physiological and pharmacological approach using ER-deficient mice, cultured vascular SMCs and ER-selective agonists. Primary cultures of vascular smooth muscle cells prepared from control and streptozotocin-injected rats were stimulated with a cytokine mixture comprising TNF-a, interleukin-1b and interferon-g for 24 h in the presence or absence of test compounds, the ER-selective agonists PPT and DPN. The increased expression of inducible NO sythase (iNOS), a classical indicator of vascular inflammation, was significantly reduced in a concentration-dependent manner by the ERa selective agonist PPT, while the ERb selective ligand DPN further enhanced iNOS expression. To explore potential mechanisms of the distinct iNOS regulation mediated by ERa and ERb and the observed loss of regulation through ERa in SMCs from diabetic rats, we transfected both ER isoforms at increasing plasmide:reporter ratios into SMCs and measured ERE-TATA-luc activity after E2 treatment. ERa and ERb transcriptional activity in response to 1 nmol/L E2 treatment for 24 h consistently increased in both SMC groups. This suggests that the transcriptional machinery of ER isoforms is maintained in SMCs from diabetic rats. The opposite regulation of iNOS function by ER-selective agonist in control SMCs could be secondary to differential modulation of nuclear p65 translocation, an important component of E2 anti-inflammatory action. Whereas LPS enhanced the nuclear staining of p65, PPT and DPN blocked this pathway, suggesting that ER-mediated iNOS control in vascular SMCs was independent from the NF-kB system. Fasting serum glucose levels were higher in ERa -/- compared with ERb -/- or wild-type (wt) mice. Diabetes was induced in 8-week-old mice by a single i.p. injection of STZ (150 mg/Kg). Despite similar worsening of glycaemic control, exposure to STZ caused significantly greater mortality in ER? -/- (41%) than in ER? -/- (12%) or wt (14%) mice. Vascular biology studies were performed in cultured aortic rings isolated from wt and ER-knockout mice. Aortic rings were stimulated with inflammatory agents in the presence and absence of test compounds. Treatment of isolated mouse aortic rings with 1 nM 17b-estradiol for 24 h attenuated cytokine-driven formation of inducible NO synthase (iNOS) as detected by Western blotting. This effect was shared by the ERa-selective agonist PPT but not by the ERb-selective agonist DPN. Accordingly, 17b-estradiol reduced iNOS formation in aortic rings from ERb -/- but not ERa -/- mice. To sum up, combined use of ER-knockout mice and ER-selective agents provided evidence for an anti-inflammatory role for ER? as well as a potential pro-inflammatory role for ER? in the arterial wall. Cellular and animal experiments indicated that ER? and ER? play a relevant role in the control of glucose metabolism, vascular inflammatory responses and diabetes progression in rodents. Based on the present findings, ER?-selective activation may represent a promising pharmacologic approach to the therapeutic modulation of diabetic vascular dysfunction.