Mos Of Age Research Articles

Lack of evidence for GWAS signals of exfoliation glaucoma working via monogenic loss-of-function mutation in the nearest gene.

Exfoliation syndrome (XFS) is a systemic disease of elastin-rich tissues involving a deposition of fibrillar exfoliative material (XFM) in the anterior chamber of the eye, which can promote glaucoma. The purpose of this study was to create mice with CRISPR/Cas9-induced variations in candidate genes identified from human genome-wide association studies (GWAS) and screen them for indices of XFS. Variants predicted to be deleterious were sought in the Agpat1, Cacna1a, Loxl1, Pomp, Rbms3, Sema6a, and Tlcd5 genes of C57BL/6J mice using CRISPR/Cas9-based gene editing. Strains were phenotyped by slit-lamp, SD-OCT imaging, and fundus exams at 1-5 mos of age. Smaller cohorts of 12-mos-old mice were also studied. Deleterious variants were identified in six targets; Pomp was recalcitrant to targeting. Multiple alleles of some targets were isolated, yielding 12 strains. Across all genotypes and ages, 277 mice were assessed by 902 slit-lamp exams, 928 SD-OCT exams, and 358 fundus exams. Homozygosity for Agpat1 or Cacna1a mutations led to early lethality; homozygosity for Loxl1 mutations led to pelvic organ prolapse, preventing aging. Loxl1 homozygotes exhibited a conjunctival phenotype of potential relevance to XFS. Multiple other genotype-specific phenotypes were variously identified. XFM was not observed in any mice. This study did not detect XFM in any of the strains. This may have been due to species-specific differences, background dependence, or insufficient aging. Alternatively, it is possible that the current candidates, selected based on proximity to GWAS signals, are not effectors acting via monogenic loss-of-function mechanisms.

Male Offspring Exposed to Maternal Hyperandrogenemia Have Exaggerated Pressor Response to Angiotensin II That Is Partially Attenuated by Etanercept

Background: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy and major cause of hyperandrogenemia in women. Our group has previously shown that male offspring (F1) exposed to maternal hyperandrogenemia are born smaller (indicative of intra-uterine growth restriction) and have exaggerated pressor response to angiotensin (Ang) II as adults. Tumor necrosis factor-α (TNF-⍺) has been shown to play a role in Ang II-mediated hypertension in different models. Increased pressor response to Ang II could signal increased risk of cardiovascular diseases later in life. Therefore, the present study was designed to test the hypothesis that exposure to maternal hyperandrogenemia programs intrarenal inflammation that plays a role in the exaggerated pressor response to Ang II in male F1s. Methods: At 4 weeks (wks) of age, female Sprague Dawley (SD) rats were fed a purified diet (10% kcal from fat) and implanted (s.c.) with either 5α-dihydrotestosterone pellets (7.5 mg/90 d; hyperandrogenemic females (HAFs)) or placebo pellets (PBO), that were replaced throughout life. At 9-12 wks of age, HAFs and PBOs were paired with SD males and allowed to get pregnant, deliver and lactate their offspring (F1-HAF and F1-PBO) until weaning (3 wks of age). Urine was collected from male F1s (n = 6-10/grp, 4-5 months (mos) of age, 1 pup/litter) which were then euthanized. Kidney cortices were used for the assay of pro-inflammatory cytokines using Bioplex. Another batch of male F1s (n = 4-7/grp, 8-10 mos of age, 1 pup/litter) were implanted with radiotelemetry transmitters. After recovery, male F1s were treated with either etanercept (a TNF-⍺ antagonist; ETA; 1.2 mg/kg, twice a week s.c. injection; ETA-F1-HAF and ETA-F1-PBO grps) or vehicle (0.9% saline; VEH; VEH-F1-HAF and VEH-F1-PBO grps) for 2 wks. Mean arterial pressure (MAP) was then measured for 6 days (Pre-enalapril), followed by treatment with enalapril (25 mg/kg/d for 9 d in drinking water) then Enalapril + Ang II (150 ng/kg/min for 6 d, s.c. minipump). Results: Male F1-HAF had significantly higher renal cortical interleukin (IL)-1α, IL-1β, IL-7 and TNF-α (203.8 ± 14 vs 141.7 ± 10, 331.4 ± 27 vs 233.7 ± 13, 524.7 ± 41 vs 379.1 ± 46, and 579.8 ± 47 vs 423.7 ± 47 pg/g tissue, respectively; p<0.05 for all), proteinuria (14.1 ± 2.4 vs 8.6 ± 0.9 mg/24 h, p<0.05), urinary kidney injury molecule-1 (1058.0 ± 123 vs 557.0 ± 122 pg/24 h, p<0.05) compared to male F1-PBO. VEH-F1-HAF had exaggerated pressor response to Ang II compared to VEH-F1-PBO starting at day 3 until day 6 of Ang II (154 ± 6 vs 128 ± 6 mmHg, p<0.05) despite similar MAP pre-enalapril (116 ± 3 vs 118 ± 2 mmHg, p=NS) and after enalapril (97 ± 3 vs 96 ± 1 mmHg, p=NS). ETA-F1-HAF had significantly lower MAP compared to VEH-F1-HAF on days 3 (119 ± 10 vs 138 ± 3 mmHg, p<0.05), 4 (131 ± 9 vs 149 ± 5 mmHg, p<0.05) and 5 (142 ± 6 vs 154 ± 5 mmHg, p<0.05) of Ang II treatment. Yet, on day 6 of Ang II treatment, MAP was similar between VEH-F1-HAF and ETA-F1-HAF (154 ± 6 vs 146 ± 5 mmHg, p=NS) and significantly higher in both groups compared to ETA-F1-PBO (133 ± 12 mmHg) and VEH-F1-PBO (128 ± 6 mmHg) [p < 0.05]. Conclusion: Exaggerated pressor response to Ang II in male F1s exposed to maternal hyperandrogenemia could be partly mediated by the pro-inflammatory cytokine TNF-α. Future studies will determine the role of different immune cells in this response and the underlying epigenetic changes in male F1s exposed to maternal hyperandrogenemia. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM121334 (JFR, NMS), R01HL135089 (JFR), R01AG075963 (JFR). The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This work was also supported by the American Heart Association Career Development Award Number 938320 (NMS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phospho-mimetic cardiac troponin I Ser43/45 produces early changes in mitochondria.

Our work tests whether chronic protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) Ser43/45 diminishes cardiac function and initiates progressive heart failure via early increases in oxidative stress and mitochondria. The current work focuses on early gene expression responses in mice after cardiac-specific phospho-mimetic cTnI S43/45D (SD) replacement of endogenous cTnI. Dose-dependent changes in cTnISD replacement do not significantly change function, when measured by echocardiography at 1 month (mos) of age in male or female mice. However, by 2 mos of age, the highest cTnISD replacement line develop significant dysfunction and 100% mortality by 16 weeks of age, with males (M) dying earlier than females (F; F = 14.6 ± 0.4 wks, n = 32; M = 9.3 ± 0.3 wks, n = 68). Based on gene expression measured by quantitative PCR (QPCR) analysis, 1 mos old hearts from this high replacement cTnISD line of mice develop significant reductions in mitochondrially-encoded cytochrome oxidase and cytochrome b in males, which are followed by similar changes at 2 mos of age in females. Reductions in gene expression of critical mitochondrial proteins PGC-1α and SOD2 also develop by 1 mos of age in males. In contrast, there are no detected changes in gene expression of the endoplasmic reticulum (ER) and nuclear stress markers, ATF6 and Nrf2, respectively for males and/or females by 2 mos of age in the high replacement cTnISD mice. Ongoing work is determining whether similar changes develop in the protein expression of these genes in young adult mice. The present results are consistent with the idea that chronic modification of the sarcomere produces a stress signal leading to early mitochondrial and oxidative stress responses, which are predicted to lay the foundation for the progressive deterioration in cardiac function observed in this model.

Abstract P184: Aging Male Offspring Of Polycystic Ovary Syndrome Rat Model Have Normal Blood Pressure And Exaggerated Pressor Response To Angiotensin II

Background: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia and elevated blood pressure (BP). Male offspring of hyperandrogenemic female (HAF) rats, PCOS model, have low birth weight, with normal BP, but exaggerated pressor response to Angiotensin (Ang) II as adults. The present study tested the hypothesis that with aging, male HAF offspring are at increased risk of developing hypertension (HT). Methods: Hyperandrogenemia was induced in female SD rats (5α-dihydrotestosterone pellets 7.5 mg/90 d, s.c. at 4 wks of age and throughout life). HAF and controls (10-12 wks of age) were mated, allowed to deliver and lactate. HAF and control male offspring (F1 HAF and F1 Contr. ) were left untreated until 16-20 mos of age. Body composition (echoMRI) and proteinuria were measured in aging offspring. BP was measured (baseline; 9 d, enalapril (25 mg/kg/d); 7d) by telemetry. Rats were then given Ang II (50 ng/kg/min, s.c. minipumps) or saline (No Ang II) for 13 d. High salt (4%) diet (HSD) was started for both Ang II and No Ang II groups on day 8 of Ang II. Results: Aging male F1 HAF had similar fat mass, but lower lean mass (433.7 ± 6 vs 453.6 ± 7 g, p< 0.05) and body weight (552.3 ± 11 vs 590.4 ± 11 g, n = 8-16, p<0.05) than F1 Contr. . Despite higher proteinuria in F1 HAF (412 ± 42 mg/24h vs 292 ± 47 mg/24h, n = 11-13, p<0.05), baseline mean arterial pressure (MAP) was similar between F1 HAF and F1 Contr. (130 ± 1 mmHg vs 126 ± 4 mmHg, respectively, n = 8, p=NS). Enalapril decreased MAP similarly in both F1 HAF and F1 Contr. (110 ± 2 mmHg vs 107 ± 3 mmHg, respectively). On low salt, Ang II increased MAP to higher levels in F1 HAF than F1 contr., saline-treated>F1 HAF and F1 Contr. (140 ± 11 mmHg vs 119 ± 10 mmHg, 105 ± 3 mmHg and 103 ± 4 mmHg, respectively, n = 4, p<0.05 F 1 HAF vs other grps). With 6 days of HSD, MAP was similar between F1 HAF and F1 Contr. not treated with Ang II (142 ± 6 mmHg and 132 ± 9 mmHg, respectively). MAP was also similar between F1 HAF and F1 Contr. with Ang II (169 ± 1 mmHg and 159 ± 9 mmHg, respectively). However, both Ang II-treated groups had significantly higher MAP compared to their respective No Ang II control groups. Conclusion: Aging male HAF offspring do not develop hypertension, but are at increased risk of renal injury and cardiovascular disease due to enhanced pressor sensitivity to Ang II.

PSI-32 Effect of pulmonary arterial pressure on reproductive performance in Angus heifers located in south central Wyoming

Abstract Pulmonary hypertension (i.e., high altitude disease) occurs in locations above 1,600 m in elevation and causes heart failure and death. Pulmonary arterial pressure scoring is an effective indicator of pulmonary hypertension and high-altitude disease. The objective of this study was to look at the effect of pulmonary arterial pressure (PAP) scoring on reproductive performance in Angus heifers. First service conception (FSC), pregnancy rate, and pulmonary arterial pressure scores were obtained from the Colorado State University Beef Improvement Center for 2,503 Angus heifers between 1995 and 2016. All spring-calving females were exposed to estrous synchronization and one-round of artificial insemination. After a 10-d holding period heifers were exposed to natural service sires for 60 days. First service conception and pregnancy rate traits were determined using ultrasonography via rectal palpation at 30 and 60 days post artificial insemination. Pulmonary arterial pressure scores were collected at ~12 mos of age. Data were analyzed with ANOVA as a completely randomized block design, blocking by birth year. Fixed effect was pulmonary arterial pressure group [high (>42mm Hg) vs. low (<42 mmHg)]. Logistic regression was used to further investigate fertility traits with PAP scores. Nodifferences were detected among pulmonary arterial pressure group for first service conception (P > 0.05); however, high pulmonary arterial pressure heifers had lower pregnancy rates compared to low PAP heifers (0.86 vs. 0.91 ± 0.01; P = 0.03). Logistic regression supported these findings, identifying PAP score as a significant predictor of breeding season pregnancy rate (P < 0.05), indicating an inverse relationship between PAP score and pregnancy rate. Cumulatively, results show an unfavorable relationship between heifers with high pulmonary arterial pressure scores and overall pregnancy rate.

Abstract 018: Female Offspring of Polycystic Ovary Syndrome Rat Model Exhibit Insulin Resistance and Tendency Toward Increased Blood Pressure With Aging

Background: Polycystic ovary syndrome (PCOS) is associated with metabolic dysfunction and elevated blood pressure (BP). Whether daughters born to PCOS mothers exhibit a PCOS phenotype is controversial. In addition, whether daughters of PCOS women develop metabolic dysfunction and elevated BP with aging has not been reported. Objectives: The present study tested the hypothesis that aging causes an increase in MAP and metabolic dysfunction in female offspring of PCOS rats (F1 DHT ) along with changes in matrix metalloproteinase-9 (MMP-9) and transforming growth factor-β (TGF-β). Methods: PCOS was induced in SD females by implantation of 5α-dihydrotestosterone pellets (DHT; 7.5 mg/90 days, sc) starting at 4 wks of age. Rats were mated with SD males, and allowed to deliver and lactate. F1 DHT were left untreated and were studied at 14-16 wks or 16-18 mos of age. Control dams were implanted with placebo pellets sc, allowed to mate, deliver and suckle their offspring (F1 PL ). Results: MAP (radiotelemetry) in 14-16 wk F1 DHT females was not different from F1 PL, nor did they exhibit other PCOS characteristics. With aging, F1 DHT rats showed a strong tendency for increased MAP compared to F1 PL (107 ± 4 versus 93 ± 1 mmHg; p = 0.07 (n=2-4)). Plasma MMP-9 and TGF- β1, 2 and 3 were not different between groups. Fasting blood glucose was not different between groups (91 ± 2.6 vs 90 ± 1.6 mg/dl), but F1 DHT rats showed significant hyperinsulinemia (0.9 ± 0.05 vs 0.78 ± 0.03 ng/ml, p ≤ 0.05) and insulin resistance (increased HOMA-IR index; 3.3 ± 0.2 vs 2.8 ± 0.1, p ≤ 0.05, (n = 5-6)). Lean mass (248.3 ± 5.5 vs 241.2 ± 2.5 g), fat mass (38.7 ± 4.8 vs 39.7 ± 2.2 g) and total water (185.8 ± 4.8 vs 179.9 ± 3 g) by Echo MRI, in addition to body weight (310.7 ± 7.7 vs 298.1 ± 4.6 g ; (n = 6-8)) were also not different between groups. Conclusion: MAP in aging female offspring of PCOS rat model tends to be increased along with hyperinsulinemia and insulin resistance with no changes in fasting glucose, lean/fat mass, body weight, MMP9 or TGF-β. The data suggest that women whose mothers had PCOS may be at increased risk for hypertension and metabolic dysfunction with aging, although the mechanisms for the hypertension are not clear.

DNA Methylation at Birth Is Associated with Adiposity and Infant Growth during the First Six Months of Life in the Healthy Start Study

In utero exposures have been shown to impart increased risk for obesity in the offspring and are associated with epigenetic changes. DNA methylation was measured in 351 umbilical cord blood (UCB) samples within the Healthy Start Study, a well-characterized prebirth cohort that is following mother-infant pairs from pregnancy-early childhood. The primary objective was to test if DNA methylation at birth was associated with fat mass (FM) gains and infant growth (weight-for-age Z-score, WAZ) during the first 6 months (mos) of life to identify potential biomarkers of future obesity risk. DNA methylation was assessed in UCB via the Illumina 450K array. Adiposity was measured at birth and 4-6 mos of age via PeaPod. Infant weight was obtained from medical records. After adjusting for cellular heterogeneity, sex, and race we found 742 methylation sites (CpGs) through a univariable analysis that were associated with the rate of FM (g/day) in the first 6 mos of life (p < 0.001). Then, partial least squares (PLS) fitting using a multivariable analysis selected a subset of 235 predictor CpGs in this list for KEGG pathway analysis. Similarly, 1314 CpGs were associated with WAZ (p < 0.001); PLS selected a subset of 198 CpGs for KEGG analysis. Using a FDR < 0.001 for KEGG analysis, 3 pathways were associated with FM gain (Metabolic pathways, Glyoxylate and dicarboxylate metabolism, Neurotrophin signaling) and 8 pathways for WAZ gain (Gastric acid secretion, Oocyte meiosis, Metabolic pathways, Endocytosis, Tight Junction, Oxytocin signaling, GnRH signaling, and Inflammatory mediator regulation of TRP channels). The KEGG: Metabolic pathways was identified for both FM and WAZ gain, and 3 gene targets enriched in this pathway, ATP5C1, CYP26A1, and IDH3A are known to be involved in fat metabolism. Our studies have identified potential candidate methylation biomarkers, present at birth, which can be used for early risk detection in order to mitigate development of childhood obesity. Disclosure S.J. Borengasser: None. A.P. Starling: None. W. Zhang: None. J. Friedman: Advisory Panel; Self; Janssen Pharmaceuticals, Inc.. R.F. Hamman: None. I. Yang: None. K. Kechris: None. D. Dabelea: None.

Abstract P150: Sex Difference of Hypertension in Spontaneously Hypertensive Rats: Role of Mitochondrial Oxidative Stress

Men have higher blood pressure (BP) than premenopausal women. Pressor response to oxidative stress may be a major contributor to the sex difference in BP control. Mitochondrial oxidative stress is associated with hypertension; however, whether mitochondrial oxidative stress plays a role in the sex difference in BP is unknown. In the present study, we tested the hypothesis that mitochondrial oxidative stress contributes to the sex difference in BP regulation in spontaneously hypertensive rats (SHR). Young intact (iYMSHR) and castrated males (cYMSHR), and females SHR (YFSHR) (3 mos of age) were implanted with radiotelemeters, and after a 4 day baseline BP, were treated with mitoTempo (0.75 mg/kg/d, sc minipumps), a specific scavenger of mitochondrial superoxide, for 7 days. Following 10 days washout of mito-tempo, rats were treated with Tempol (30 mg/kg/day, po drinking water) for 7 days. iYMSHR have higher blood pressure (by telemetry) than cYMSHR and YFSHR (148±1 mmHg, n=5, vs 132±1 mmHg, n=5, and 139±1 mmHg, n=5; p<0.01, respectively). MitoTempo reduced BP by 6% in iYMSHR (147±1 vs 139±1, n=5; p<0.05) compared to females (3%: 139±1 vs 136±1; n=5; p: NS) and castrated males (4.5%: 132±1 vs 126±1, n=5; p<0.05). After 10 days washout, tempol reduced BP only in iYMSHR (144±1 vs 130±1 mmHg, n=5; p<0.05). Our results suggest that mitochondrial oxidative stress may contribute to BP regulation in male SHR, but has no effect in females. The data also suggest that the presence of testosterone is necessary for the pressor response to oxidative stress in males since Tempol had no effect on BP in castrated males. Further studies examining the effect of steroid hormones and mitochondria in BP regulation are necessary to elucidate the importance of mitochondrial oxidative stress on sex difference of hypertension.

Study of Acute Kidney Injury in hyperandrogenemic female rats

Chronic hyperandrogenemia is hallmark of polycystic ovarian syndrome (PCOS), affecting ~10% of women of reproductive age. Compared to men, women are protected against ischemia‐ reperfusion (I/R) acute kidney injury (AKI), but the potential susceptibility of women that have elevated androgens, as women with PCOS, to develop I/R‐induced AKI is not clear. Other models of hyperandrogenemia in females in which I/R AKI was studied utilized levels of testosterone similar to males. Using an established female rat model of PCOS that has a 3 fold increase in DHT, such as women with PCOS and 10 fold lower than in males, we tested the hypothesis that chronic hyperandrogenemia increases the risk of I/R‐induced AKI compare to placebo controls. Female SD rats were implanted with dihydrotestosterone (DHT, 7.5mg/90d) or placebo pellets (n=11/grp) beginning at 6 wks of age; pellets were changed every 85 d. At 6–7 mos of age, rats (n=3–4/grp) were subjected to sham or renal I/R with bilateral clamping of renal vessels for 30 min at 37±0.5°C. Rats were placed in metabolism cages for 24hrs reperfusion. At 48–72hr post‐surgery rats were again put in metabolic cages for 24hr, and then euthanized for blood and tissue samples. I/R increased plasma creatinine (PCr) 24hr post reperfusion in placebo (2.58±0.34 vs 0.86±0.1 mg/dL, p<0.05) as well as DHT treated rats (4.53± 0.46 vs 0.9±0.06 mg/dL, P<0.001) compared to sham‐I/R group. As noted, PCr in DHT‐treated rats was significantly higher than in placebo rats (4.53± 0.46 vs 2.58±0.34 mg/dL, p<0.05). When DHT rats were pre‐treated with androgen receptor antagonist (Flutamide, 30mg/kg/day, s.c.) for three days prior to I/R, PCr levels 24hr post reperfusion were not different than DHT–treated I/R groups (4.75 0.52 vs 4.53± 0.46 mg/dL, p=ns). These data show that rat exposure to chronic hyperandrogenemia causes significantly greater renal injury in response to I/R AKI than in controls. This injury is independent of the androgen receptor suggesting other mechanisms may be contributing to the higher level of renal injury after I/R AKI in females with PCOS.Support or Funding InformationSupported by NIH R01HL66072, P01HL05971.

Impact of Prenatal Exposure to Humanized High Fat Diets on Murine Metabolic Phenotype

Prenatal exposures, including maternal diet, can alter risk for metabolic disease across the lifecourse. This study examines the effects of 2 prenatal high fat diets (HFDs) on mice offspring weight, nutrient utilization, and activity at 2 and 4 months of age. Two HFDs (43% fat kcal) were developed to mirror human intake: 1) Western (W) and 2) Mediterranean (M). Dams (n=44) were randomized to W, M, or Control (C) (7% fat) 2 wks prior to mating through pregnancy and lactation; pups were weaned onto C diet. One male (n=36) and 1 female (n=35) pup/litter were followed longitudinally with weekly weights and comprehensive phenotyping at 2 and 4 mos of age. ANOVA with Tukey's post-hoc analysis compared pups' mean metabolic outcomes across maternal prenatal diet groups. Prenatal HFD had a greater impact on females than males. M females were less ambulatory (p=0.046, 0.021) with less horizontal (p=0.023, 0.063) and vertical (p=0.035, 0.031) activity than C females at 2 and 4 mos, respectively. A similar trend was observed in 2 mo M males with less ambulatory (p=0.064) and horizontal (p=0.063) activity; by 4 mos, no activity differences were noted. At 4 mos, glucose oxidation (p=0.044) and respired CO2 (p=0.016) were lower in M females vs. C, consistent with increased utilization of fat as a primary fuel. All significant findings were during the light cycle, suggesting HFD impacts resting activity. MANOVA analysis found increased weight in M females but not males (p=0.047 and p=0.260). Thus, prenatal HFD alters female activity and nutrient utilization into early adulthood. A third round of phenotyping will be performed at 8 mos to characterize life course effects.

Infant nutritional outcomes in an integrated agriculture, health and nutrition program in Western Kenya

The Mama SASHA project integrated an orange‐fleshed sweetpotato (OFSP)‐focused, agricultural‐nutrition education intervention into delivery of routine health services and pregnant women's clubs. It aimed to improve diets, nutrition and health of pregnant /lactating women and their infants. We enrolled 505 women from intervention and control facilities during first antenatal care visit in early pregnancy. Infant anthropometry, vitamin A and iron status were assessed at 4 and 9 mos of age and anemia at 9 mos. There were no significant socioeconomic differences at enrollment. OFSP consumption was significantly higher and maternal MUAC significantly lower in intervention mothers at enrollment and throughout the follow up period. Mean birth weight was 3.27±0.5 kg and did not differ between groups. At 9 mos household food security and coverage with VA supplementation were significantly higher in controls; consumption of OFSP was significantly higher in intervention infants. From 4 to 9 mos postpartum, stunting increased significantly in controls (6.9% to 10.6%, p<0.05); stunting decreased nonsignificantly in the intervention group (11.5% to 10.3%, p>0.05). There was no change in either underweight from 4 to 9 mos (intervention: 3.1% to 3.3%; controls: 3.9% to 4.3%) or wasting (intervention: 2.1% to 1.6%; controls: 2.9% to 2.7%). There were no significant group differences in infant micronutrient status or anemia at 4 or 9 months. Further analysis will evaluate impact using mixed models that control for clustering, repeated measures and confounding.

Understanding the developmental course of the acoustic properties of the human outer and middle ear over the first 6 months of life by using a longitudinal analysis of power reflectance at ambient pressure.

As shown by tympanometric and wideband acoustic immittance (WAI) measures, developmental changes that occur in the middle ear and the external auditory canal can affect the mechano-acoustic properties of the middle ear system during the first 2 years of life. However, these observed differences and the specific course of maturation have not been fully investigated. The overall goal of the current study was to define the time course and rate at which functional maturation of the middle ear occurs in human infants through wideband acoustic immittance (WAI). It was also within the scope of this study to establish normative data to characterize acoustic properties of the normal middle ear in infants from birth to 6 mo of age. Newborns were tested longitudinally at 1-mo intervals up to 6 mo of age for a total of six visits. All babies passed transient evoked otoacoustic emission screening on each visit. Tympanograms and WAI were recorded, and the distributions of WAI patterns were analyzed as a function of age. WAI was measured across a wide range of frequencies from birth to 6 mos of age in a cohort of 31 infants. Test-retest differences for WAI were also evaluated. Thirty-one newborns were recruited. A repeated-measure analysis of variance was conducted to investigate whether the variations observed in power reflectance at ambient pressure across the six visits were statistically different. All significant findings were subject to Greenhouse-Geisser correction for repeated-measures sphericity and inflated type-I error. RESULTS showed that power reflectance increased (closer to 1) at low frequencies (<400 Hz) and decreased (closer to 0) at high frequencies (>2000 Hz) as a function of age. There was very little change in power reflectance from 600 to 1600 Hz across the first 6 mo of life. Most group changes that were observed were also consistently shown in longitudinal changes observed within individual subjects. The overall maturation of the middle ear can result in a lower reflectance at higher frequencies and a higher reflectance at lower frequencies. This could be explained by known changes in ear-canal and/or middle-ear structures across this time period, which could lead to changes in mass and resistance. An increase in volume in the middle-ear cavity, reduction of middle-ear debris, and overall decrease in resistive elements might be contributing to these changes. Significant differences were observed between each visit, which could warrant the use of age-specific norms when applying WAI data to infants less than 6 mo of age.

Peripheral blood mononuclear cell (PBMC) gene expression in human infants shifts from innate to adaptive immunity in the first six months of life (247.5)

The newborn immune system is developmentally naïve, but undergoes rapid maturation upon exposure to environmental antigens. As part of a clinical trial in infants aimed at assessing how diet impacts PBMC transcriptome, genes that were developmentally regulated independent of diet‐ were discovered. Blood samples were obtained from breast‐fed (n=60) and three sets of formula‐fed (n=186 total) infants at 1, 4 and 6 mos of age. PBMC were isolated, RNA extracted and gene expression assessed using the Affymetrix HuGene 2.0ST gene chip. To evaluate patterns of gene expression, Weighted Gene Correlation Network Analysis (WGCNA) was performed on 6,441 probe sets with an overall 4x3 ANOVA FDR p‐value&lt;0.3. Three gene clusters with similar patterns of expression in each diet (1 vs. 4 &amp; 6 mos) encompassing 1085 genes were identified. Functional analysis was performed with MetaCore. Pathways with higher expression at 1‐mo reflected cellular proliferation, innate defense processes (defensins, hydrogen peroxide‐induced killing) and Th17‐derived cytokines. By 4‐ to 6‐mos of age, genes associated with humoral immunoglobulin‐mediated immunity and interferon and IL‐4 signaling predominated. These data illustrate for the first time the developmental ontogeny of gene expression in circulating immune cells of human infants.Grant Funding Source: Arla Foods and Biostime

Effects of exercise and body temperature on eNOS, SIRT1, SIRT3 and Hsp70 expression in rat plantaris muscles (1164.6)

Previously, we have demonstrated a dramatic increase in mitochondrial biogenesis in skeletal muscle of rats exercise trained while maintaining a constant core temperature. In this study, we explored the role of body temperature during exercise on the expression of multiple proteins (eNOS, SIRT1, SIRT3, and Hsp70) believed to play a role in mitochondrial biogenesis. Female, Sprague‐Dawley rats (5 mos of age) were divided into three groups sedentary (S), exercise in 22°C room (ET), and exercise while maintaining core temperature (E). Exercised animals trained for 5 weeks on a motor‐driven treadmill at 30 m/min, 60 min/day, 5 days/wk during final 2 wks. Core temperature was held constant in E by reducing room temp to 6‐8°C. Plantaris cytochrome oxidase activity (μmoles O2/min/g wet wt) was significantly (P&lt;0.05) elevated in both ET (37.00±2.10) and E (47.14±4.11) versus S (24.15±1.37) and significantly greater in E versus ET. Preliminary results reveal no significant differences in SIRT1, SIRT3, or eNOS expression in the soleus muscles of S, E, or ET. Hsp70 was significantly (P&lt;0.05) elevated in the ET group. These data suggest that although mitochondrial biogenesis increases in this model, eNOS, SIRT1 and SIRT3 do not parallel this increase and may not play a role in this process.

Osteopontin supplementation of formula shifts the peripheral blood mononuclear cell transcriptome to be more similar to breastfed infants (38.3)

OPN is a key modulator in cell‐mediated immune and anti‐inflammatory responses that is present in breast milk, but not infant formula. A randomized clinical trial was conducted to study the impact of bovine OPN in formula on infant PBMC gene expression. Mothers chose to either breast‐(BF) or formula‐feed their infant. Formula‐fed infants received: formula (FF), formula with 65 mg/L (F65) or 130 mg/L (F130) OPN. Blood samples were collected at 1, 4 and 6 mos of age, PBMC were isolated, RNA extracted and gene expression assessed using the Affymetrix HuGene 2.0ST gene chip. To evaluate patterns of gene expression, Weighted Gene Correlation Network Analysis (WGCNA) was performed on 6,441 probe sets with an overall 4x3 ANOVA FDR p‐value&lt;0.3. Three modules (4066 probe sets) identified patterns where FF65 infants were similar to BF and different from FF and F130 across time. Functional analysis was performed with MetaCore. Pathways with higher expression in BF and F65 focused on cell proliferation and cell‐cell adhesion, supporting increased production and maturation of immune cells. In contrast, PBMC from FF and F130 infants expressed more genes related to immunoglobulin synthesis and receptors, including a gene associated with risk of childhood allergy. Thus, supplementing infant formula with OPN shifted PBMC gene expression to be more similar to BF and may support improved immune development.Grant Funding Source: Supported by: Arla Foods Ingredients and Biostime

Abstract 61: Hypertension in a Rat Model of Postmenopausal Hyperandrogenemia (Polycystic Ovary Syndrome): Roles of the Renin-Angiotensin and Sympathetic Nervous Systems

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in premenopausal women. PCOS is characterized by hyperandrogenemia that does not abate after menopause. Hyperandrogenemic postmenopausal women (HA-PMW) are hypertensive, but the mechanisms responsible have not been elucidated. Hypertension in PMW is less well controlled than in age-matched men. We have developed a model, the hyperandrogenemic postcycling female (PMHAF) rat, aged 13-14 mos, that exhibits many of the characteristics found in HA-PMW. In the present studies we tested the hypothesis that the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) contribute to hypertension in PMHAF rats. Female Sprague-Dawley rats were implanted with dihydrotestosterone (7.5mg/90d) or placebo pellets beginning at 4 wks of age, and pellets were changed every 85 d. At 12-13 mos of age, PMHAF rats were obese compared to placebo controls, and CT scans showed that both total adipose (TAF) and visceral adipose (VAF) were increased in PMHAF vs placebo controls (n=4/grp) (TAF: 7.9±0.5 vs 5.46±1.00 AU/g BW, p&lt;0.05; VAF, 4.60±0.30 vs 3.45±0.07 AU/g BW, p&lt;0.05). PMHAF rats (n=3/grp) were implanted with radiotelemetry transmitters, and 2 wks later, baseline MAP was measured for 5 d. Then rats were given losartan (40mg/kg/d) or tap water placebo for 5 days, and then tap water was returned. After additional 5 d and return of MAP to baseline, rats were implanted with minipumps containing terazocin and propranolol (10 mg/kg/d for each drug, sc, for 7 d) or saline vehicle. Baseline MAP in PMHAF rats was (128±9 vs 105±9 mmHg, p&lt;0.05). Losartan reduced MAP more in PMHAF than placebo controls (111±9 vs 95±7 mmHg, p&lt;0.05). Intrarenal angiotensinogen and ACE1 mRNA levels were increased by 8 fold and 2 fold, respectively, in PMHAF rats vs placebo controls (p&lt;0.05). α 1 -β 1,2 -Adrenergic blockade reduced MAP by 13% in PMHAF rats, the same level as in placebo controls, and had no effect in placebo controls (110±12 vs 102±5 mmHg, p&lt;0.05 vs baseline MAP). These data suggest that both the RAS and SNS are upregulated in PMHAF rats and contribute to their hypertension. These data also suggest that multiple drug therapy is likely to be necessary to control MAP in PMW with hyperandrogenemia. Supported by PO1HL51971.

Abstract 251: 20-HETE Contributes to the Hypertension in a Model of Postmenopausal Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in premenopausal women. PCOS is characterized by hyperandrogenemia that does not abate after menopause. Hyperandrogenemic postmenopausal women (PMW) are hypertensive, but the mechanisms responsible have not been elucidated. Hypertension (HT) in PMW is less well controlled than in age-matched men, which suggests that different mechanisms may be mediating the HT in PMW. We have developed a model, the hyperandrogenemic postcycling female (PMHAF) rat, aged 13-14 mos, that exhibits many of the characteristics found in hyperandrogenemic PMW. In the present study we tested the hypothesis that 20-HETE contributes to the HT in PMHAF rats. Female Sprague-Dawley rats were implanted with dihydrotestosterone (7.5mg/90d) or placebo pellets beginning at 4 wks of age, and pellets were changed every 85 d. At 13-14 mos of age, preglomerular renal microvessels were isolated from PMHAF and placebo control rats (n=6/grp) and 20-HETE and ω-hydroxylase activity were measured by LC-MS. mRNA for CYP4A enzymes were measured by real time RT-PCR. Basal 20-HETE levels in the renal vasculature increased by 60% in PMHAF vs placebo controls (0.398±0.22 vs 0.245 ±0.68 pmol/mg; p&lt;0.05) and 20-HETE synthesis from arachidonic acid was increased by 17-fold (19.6±5.1 vs 1.18±0.14 pmol/min/mg, p&lt;0.001). CYP4A2 mRNA levels were increased 15-fold and CYP4A8 mRNA levels were decreased by 60% in PMHAF rats compared to placebo controls (n=6/grp; p&lt;0.05, PMHAF compared to placebo controls). In another group of rats, acute infusion of an inhibitor of 20-HETE synthesis, HET-0016 (10 mg/kg bolus and 1 mg/kg/h iv) reduced MAP by about 20 mm Hg in PMHAF rats. These data suggest that synthesis of 20-HETE in the vasculature is upregulated in PMHAF rats and contributes to their hypertension. The studies also suggest that novel mechanisms may be responsible for hypertension in PMW with hyperandrogenemia compared to age-matched men, and as such, novel therapeutic approaches may be necessary to control their blood pressures. Supported by PO1HL51971.

Anti-aging Effects of Co-enzyme Q10 on Periodontal Tissues

Oxidative stress is associated with age-related reactions. The anti-oxidative effects of a reduced form of co-enzyme Q10 (rCoQ10) suppress oxidative stress, which may contribute to the prevention of age-related inflammatory reactions. We examined the effects of topically applied rCoQ10 on periodontal inflammatory reactions in a rat aging model. Male Fischer 344 rats, 2 (n = 6) and 4 mos (n = 18) of age, were used. All of the two-month-old rats and 6 of the four-month-old rats were sacrificed and 12 remaining four-month-old rats received topically applied ointment with or without 1% rCoQ10 on the gingival surface until they reached 6 mos of age. The rats showed an age-dependent increase in circulating oxidative stress. RCoQ10 decreased oxidative DNA damage and tartrate-resistant acid-phosphatase-positive osteoclasts in the periodontal tissue at 6 mos of age as compared with the control. The same conditions lowered gene expression of caspase-1 and interleukin-1β in the periodontal tissue. Furthermore, Nod-like receptor protein 3 inflammasomes were less activated in periodontal tissues from rCoQ10-treated rats as compared with the control rats. Our results suggest that rCoQ10 suppresses age-related inflammatory reactions and osteoclast differentiation by inhibiting oxidative stress.

Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Abstract 117Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS).To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia.Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure.It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures:No relevant conflicts of interest to declare.

Vital Capacity in Spinal Muscular Atrophy

This work describes and correlates plateau/maximum observed vital capacity (VC) with spinal muscular atrophy (SMA) severity and prognosis for autonomous breathing. SMA severity was correlated with VC, onset, paradoxical breathing, age at definitive dependence on continuous mechanical ventilation (DDCV), and age at first respiratory hospitalization and gastrostomy. Ten severe SMA 1A patients with DDCV before 6 mos of age had maximum observed and plateau VC of 100 ml or less, with plateaus for six at 10.3 (range, 3-48) mos. Another 120 had typical SMA 1B defined by any three of the following: acute respiratory failure before 12 mos of age, gastrostomy before 12 mos of age, DDCV before 10 yrs of age, and VC not exceeding 200 ml, with plateaus for 15 at 26.4 (6-138) mos. Fifteen were with mild type 1C defined by three of the following: VC exceeding 200 ml, acute respiratory failure after 1 yr of age, gastrostomy after 1 yr of age, and no DDCV before 10 yrs of age and had a plateau/maximum observed mean VC of 409 (range, 200-1175) ml at 8.9 (range, 7-10) yrs of age. Of 88 patients with SMA 2 and paradoxical breathing (2A), 16 had a mean plateau/maximum observed VC of 758 (range, 460-2100) ml. DDCV with no autonomous breathing always followed plateauing of VC. Types 2B, 3, and 4 patients attained normal VC. Intergroup VC differences were significant (P < 0.05) for 1A, 1B, 1C, 2A, and 2B-4. When intubated and "unweanable," 50-ml VC signaled the ability for autonomous breathing 1 to 21 days after extubation. VC should be monitored from birth. It correlates with prognosis with SMA 1A VCs not exceeding 100 ml or 1B 200 ml. Patients who attained 200 ml at any time (milder 1C) retain some ability to breathe after 10 yrs of age.