Cynthia J. Lees

Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3–5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

Effect of treatment for 3 months with human parathyroid hormone 1–34 peptide in ovariectomized cynomolgus monkeys (Macaca fascicularis).

A potential negative side effect of intermittent parathyroid hormone (PTH) therapy to treat osteoporosis is the loss of cortical bone concomitant with increased cancellous bone mass. We addressed this issue by studying the effects of PTH on whole-body, axial, and appendicular bone mass in an animal model with haversian cortical bone remodeling. Ovariectomized, young adult female cynomolgus monkeys were assigned to placebo (n = 9) or PTH groups (n = 10). The PTH group received 10 microg/kg synthetic human PTH(1-34) peptide by subcutaneous injection, 3 days/week for 6 months, and the placebo group received vehicle. Multiple endpoints of bone mass, strength, and turnover in the axial and appendicular skeleton were assessed, including dual-energy X-ray absorptiometry (DEXA), quantitative computed tomography (qCT), analysis of serum (calcium, phosphorus, alkaline phosphatase, osteocalcin, and tartrate-resistant acid phosphatase) and urinary (calcium and creatinine) biomarkers, histomorphometry, and biomechanical testing. Compared with placebo-treated animals, PTH-treated monkeys had no change in whole-body bone mass, but a 6.7% increase in spinal areal bone mineral density (aBMD) was observed. Cortical bone mass measured by qCT at appendicular sites was not affected by PTH treatment, but there were significant increases in cancellous bone mass in the proximal tibia, and a similar trend in the distal radius. Small, transient increases in serum and urinary calcium were observed, but there were no treatment-related effects on other biochemical endpoints. Increased bone formation rate (BFR/BV) in the midradius and midfemur was accompanied by a nonsignificant increase in midfemur porosity. Increased vertebral cancellous bone volume (BV/TV) was associated with greater trabecular and interstitial thickness with no effect on wall thickness. Increases in bone strength were observed in both axial (vertebral maximum stress and load at fracture) and appendicular (femoral neck fracture load) skeleton. Together, these results indicate that PTH therapy in the cynomolgus monkey results in a net gain of spinal and appendicular cancellous bone mass with no adverse effect on cortical bone.

Flagellin-F1-V Fusion Protein Is an Effective Plague Vaccine in Mice and Two Species of Nonhuman Primates

ABSTRACT A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4°C and at least 168 days at 25°C. At between 29 and 84 days at 37°C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.

Decreased Bone Mass and Strength in Ovariectomized Cynomolgus Monkeys ( Macaca fascicularis )

Agents for prevention or treatment of osteoporosis must now be tested in a large animal species that exhibits bone remodeling. Ovariectomized, nonhuman primates provide one such model, and they consistently develop osteopenia accompanied by high bone turnover rates. The goal of this study was to further characterize this model, and particularly to determine the effect of ovariectomy on bone strength in vertebrae and femoral necks. Longitudinal evaluations of spinal bone mass and serum markers of bone turnover were performed in 19 sham-ovariectomized (SHAM) and 18 ovariectomized (OVX), domestically reared cynomolgus monkeys, aged >9 years. OVX monkeys lost bone relative to both baseline values and SHAM controls. Serum markers of bone turnover were increased by OVX. After 72 weeks, both vertebral bone compressive strength and femoral neck breaking strength were significantly decreased in OVX animals compared with SHAM. Ovariectomized cynomolgus monkeys, like postmenopausal women, develop accelerated bone loss, increased bone turnover, and reduced bone strength, and provide a suitable large animal model for efficacy studies with agents for prevention or treatment of osteoporosis.

Characterization of adult prostatic progenitor/stem cells exhibiting self-renewal and multilineage differentiation.

Demonstration of the hallmarks of stem cells, self‐renewal and multilineage differentiation, is a challenge that has not been met for numerous tissues postulated to possess adult stem cells, including prostate tissue. Using a defined medium, we reproducibly isolated and maintained adult mouse prostatic cells with characteristics of progenitor/stem cells. Clonal populations of cells demonstrated tissue‐specific multilineage differentiation by their ability to generate organized prostatic ductal structures in vivo, with luminal and basal cell layers, when grafted under the renal capsules of mice in the presence of fetal rat urogenital mesenchyme. Complete differentiation was demonstrated by the expression and secretion of terminally differentiated prostatic secretory products into the lumens. Self‐renewal was demonstrated by serial transplantation of clonal populations that generated fully differentiated ductal structures in vivo. In vitro, undifferentiated cells expressed markers associated with prostate stem cells, including Sca 1 and CD49f, as well as basal cell markers (p63 and cytokeratins 5 and 14) and, at a low level, luminal cell markers (androgen receptor and cytokeratins 8 and 18). When grafted and allowed to differentiate in the presence of fetal urogenital mesenchyme, the cells differentiated into luminal cells and basal cells with more restricted protein expression patterns. These studies are the first to report a reproducible system to assess adult prostatic progenitor/stem cells.

Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys

Abstract Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.

Impairment of ovarian function and associated health-related abnormalities are attributable to low social status in premenopausal monkeys and not mitigated by a high-isoflavone soy diet

BACKGROUND Psychological stress may impair premenopausal ovarian function and contribute to risk for chronic disease. Soy isoflavones may also influence ovarian function and affect health. Here, we report the effects of a psychological stressor (subordinate social status) and dietary soy on reproductive function and related health indices in female monkeys. We hypothesized that reproductive compromise and adverse health outcomes would be induced in subordinate when compared with dominant monkeys and be mitigated by exposure to soy. METHODS Subjects were 95 adult cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six. Animals consumed a soy-free, animal protein-based diet during an 8-month Baseline phase and then, during a 32-month Treatment phase, consumed either the baseline diet or an identical diet that substituted high-isoflavone soy protein for animal protein. RESULTS Across more than 1200 menstrual cycles, subordinate monkeys consistently exhibited ovarian impairment [increased cycle length (P < 0.02) and variability (P < 0.02) and reduced levels of progesterone (P < 0.04) and estradiol (P < 0.04)]. Subordinate status was confirmed behaviorally and was associated with elevated cortisol (P < 0.04) and relative osteopenia (P < 0.05). Consumption of the soy diet had no significant effects. CONCLUSIONS (i) Psychological stress adversely affects ovarian function and related health indices in a well-accepted animal model of womens health; (ii) Similar effects may extend to women experiencing reproductive impairment of psychogenic origin; (iii) soy protein and isoflavones neither exacerbate nor mitigate the effects of an adverse psychosocial environment; and (iv) this study was limited by an inability to investigate the genetic and developmental determinants of social status.

Effects of raloxifene on bone density, biomarkers, and histomorphometric and biomechanical measures in ovariectomized cynomolgus monkeys.

ObjectiveThe purpose of this study was to determine the effect of raloxifene on bone density, strength, metabolism, and histomorphometric characteristics in ovariectomized cynomolgus monkeys. DesignA prospective, longitudinal study was designed to examine the effects of conjugated equine estrogens (0.04 mg/kg, CEE) and raloxifene (1 or 5 mg/kg, R1 and R5, respectively) on bone density, biomarkers, histomorphometry, and strength. Control groups included ovariectomized and sham-operated monkeys. Treatment was initiated the day after ovariectomy and continued for 24 months. Bone biomarker data were collected at baseline and every 3 months after surgery. Bone mass was determined at baseline and every 6 months after ovariectomy. Iliac biopsies were collected at baseline and 16 months postovariectomy, and the second lumbar vertebra and left midshaft femur collected at necropsy were examined histomorphometrically. Bone biomechanical properties were determined for the right femur and vertebrae. ResultsCompared with the placebo-treated ovariectomized monkeys, the high-dose raloxifene group had lower levels of alkaline phosphatase, tartrate-resistant acid phosphatase, urinary CrossLaps (collagen degradation products), and greater bone mass in the lumbar vertebrae. In the endocortical compartment, the high-dose raloxifene group had significantly lower mineralizing surface, mineral apposition rate, and bone formation rate in the iliac biopsy collected at 16 months and lower bone formation rate in the second lumbar vertebra. Within the midshaft femur, low-dose raloxifene significantly decreased the osteonal and total bone formation rates and also prevented the decrease in Youngs modulus induced by ovariectomy in the midshaft femur. ConclusionsHigh-dose raloxifene prevented the development of osteopenia in the ovariectomized monkey by reducing bone turnover, albeit to a lesser extent than CEE. Histomorphometric and biomarker data suggest that mechanisms underlying the effect of raloxifene differ somewhat from that of CEE.

Soy Protein Isolate Diet Does Not Prevent Increased Cortical Bone Turnover in Ovariectomized Macaques

Summary: Forty-one ovariectomized, cynomolgus monkeys were divided into 4 groups and fed a casein and lactalbumin based diet with or without 17β-estradiol, or a soy protein based diet with or without 17β-estradiol for 7 months. Histomorphometry was done on cortical bone from the mid-shaft femur. 17β-estradiol suppressed ovariectomy-induced increases in bone formation rates, regardless of dietary protein source. Soy protein alone did not prevent increased bone turnover and on the endosteal surface, it actually increased bone turnover when compared to casein/lactalbumin fed monkeys.

Suppression of Tak1 promotes prostate tumorigenesis.

More than 30% of primary prostate cancers contain a consensus deletion of an approximately 800 kb locus on chromosome 6q15.1. The MAP3K7 gene, which encodes TGF-β activated kinase-1 (Tak1), is a putative prostate tumor suppressor gene within this region whose precise function remains obscure. In this study, we investigated the role of Tak1 in human and murine prostate cancers. In 50 well-characterized human cancer specimens, we found that Tak1 expression was progressively lost with increasing Gleason grade, both within each cancer and across all cancers. In murine prostate stem cells and Tak1-deficient prostatic epithelial cells, Tak1 loss increased proliferation, migration, and invasion. When prostate stem cells attenuated for Tak1 were engrafted with fetal urogenital mesenchyme, the histopathology of the grafts reflected the natural history of prostate cancer leading from prostatic intraepithelial neoplasia to invasive carcinoma. In the grafts containing Tak1-suppressed prostate stem cells, p38 and c-jun-NH(2)-kinase activity was attenuated and proliferation was increased. Together, our findings functionally validate the proposed tumor suppressor role of Tak1 in prostate cancer.