Syed, Farhan

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18–22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20–200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm2, P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm2, P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200–500 μm) decreased only in aged sham ( P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Abstract While female mice do not have the equivalent of a menopause, they do undergo reproductive senescence. Thus, to dissociate the effects of aging versus estrogen deficiency on age-related bone loss, we sham-operated, ovariectomized, or ovariectomized and estrogen-replaced female C57/BL6 mice at 6 months of age and followed them to age 18 to 22 months. Lumbar spines and femurs were excised for analysis, and bone marrow hematopoietic lineage negative (lin–) cells (enriched for osteoprogenitor cells) were isolated for gene expression studies. Six-month-old intact control mice were euthanized to define baseline parameters. Compared with young mice, aged/sham-operated mice had a 42% reduction in lumbar spine bone volume/total volume (BV/TV), and maintaining constant estrogen levels over life in ovariectomized/estrogen-treated mice did not prevent age-related trabecular bone loss at this site. By contrast, lifelong estrogen treatment of ovariectomized mice completely prevented the age-related reduction in cortical volumetric bone mineral density (vBMD) and thickness at the tibial diaphysis present in the aged/sham-operated mice. As compared with cells from young mice, lin– cells from aged/sham-operated mice expressed significantly higher mRNA levels for osteoblast differentiation and proliferation marker genes. These data thus demonstrate that, in mice, age-related loss of cortical bone in the appendicular skeleton, but not loss of trabecular bone in the spine, can be prevented by maintaining constant estrogen levels over life. The observed increase in osteoblastic differentiation and proliferation marker gene expression in progenitor bone marrow cells from aged versus young mice may represent a compensatory mechanism in response to ongoing bone loss. © 2010 American Society for Bone and Mineral Research.

Abstract ERα acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. Introduction: Estrogen receptor α (ERα) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ERα knock-in [NERKI]) with heterozygote ERα knockout mice and studied the resulting female ERα+/+, ERα+/NERKI, and ERα−/NERKI mice. The only ERα present in ERα−/NERKI mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ERα+/NERKI mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. Materials and Methods: BMD was measured using DXA and pQCT at 3 months of age (n = 46–48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 μg/day), or ovariectomy + estradiol (1.0 μg/day; n = 10–12/group) and restudied 60 days later. Results and Conclusions: At 3 months of age, both the ERα+/NERKI and ERα−/NERKI mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ERα−/NERKI mice were the opposite of those in ERα+/+ mice. Relative to sham mice, ovariectomized ERα−/NERKI mice gained more bone (not less, as in ERα+/+ mice), and estrogen suppressed this increase (whereas augmenting it in ERα+/+ mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ERα−/NERKI versus ERα+/+ mice. Collectively, these data show that alteration of the balance between classical and nonclassical ERα signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ERα signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.