Hostname: page-component-586b7cd67f-rcrh6 Total loading time: 0 Render date: 2024-11-26T10:10:38.417Z Has data issue: false hasContentIssue false

Placental insufficiency induces a sexually dimorphic response in the expression of cardiac growth and metabolic signalling molecules upon exposure to a postnatal western diet in guinea pigs

Published online by Cambridge University Press:  26 July 2021

Jack R.T. Darby
Affiliation:
Early Origins of Adult Health Research Group, Health and Biomedical Innovation, UniSA: Clinical and Health Sciences, University of South Australia, Adelaide, SA, Australia
Jacky Chiu
Affiliation:
Physiology and Pharmacology, Western University, London, ON, Canada
Timothy R.H. Regnault
Affiliation:
Departments of Obstetrics and Gynaecology, Western University, London, ON, Canada Physiology and Pharmacology, Western University, London, ON, Canada Children’s Health Research Institute and Lawson Health Research Institute, London, ON, Canada
Janna L. Morrison*
Affiliation:
Early Origins of Adult Health Research Group, Health and Biomedical Innovation, UniSA: Clinical and Health Sciences, University of South Australia, Adelaide, SA, Australia
*
Address for correspondence: Janna L. Morrison, PhD., ARC Future Fellow (Level 3), Early Origins of Adult Health Research Group, Health and Biomedical Innovation, UniSA Clinical and Health Sciences, University of South Australia, GPO Box 2471, Adelaide, SA, Australia. Email: [email protected]

Abstract

There is a strong relationship between low birth weight (LBW) and an increased risk of developing cardiovascular disease (CVD). In postnatal life, LBW offspring are becoming more commonly exposed to the additional independent CVD risk factors, such as an obesogenic diet. However, how an already detrimentally programmed LBW myocardium responds to a secondary insult, such as an obesogenic diet (western diet; WD), during postnatal life is ill defined. Herein, we aimed to determine in a pre-clinical guinea pig model of CVD, both the independent and interactive effects of LBW and a postnatal WD on the molecular pathways that regulate cardiac growth and metabolism. Uterine artery ablation was used to induce placental insufficiency (PI) in pregnant guinea pigs to generate LBW offspring. Normal birth weight (NBW) and LBW offspring were weaned onto either a Control diet or WD. At ˜145 days after birth (young adulthood), male and female offspring were humanely killed, the heart weighed and left ventricle tissue collected. The mRNA expression of signalling molecules involved in a pathological hypertrophic and fibrotic response was increased in the myocardium of LBW male, but not female offspring, fed a WD as was the mRNA expression of transcription factors involved in fatty acid oxidation. The mRNA expression of glucose transporters was downregulated by LBW and WD in male, but not female hearts. This study has highlighted a sexually dimorphic cardiac pathological hypertrophic and fibrotic response to the secondary insult of postnatal WD consumption in LBW offspring.

Type
Original Article
Copyright
© The Author(s), 2021. Published by Cambridge University Press in association with International Society for Developmental Origins of Health and Disease

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

*

Denotes equal contribution.

References

WHO. World Health Statistics 2017: monitoring health for the SDGs. Sustainable Development Goals. World Health Organization. 2017; 3035. https://apps.who.int/iris/handle/10665/255336. License: CC BY-NC-SA 3.0 IGO.Google Scholar
Arora, S, Stouffer, GA, Kucharska-Newton, AM, et al. Twenty year trends and sex differences in young adults hospitalized with acute myocardial infarction. Circulation. 2019; 139, 10471056.CrossRefGoogle ScholarPubMed
Howard, BV, Wylie-Rosett, J. Sugar and cardiovascular disease: a statement for healthcare professionals from the Committee on Nutrition of the Council on Nutrition, Physical Activity, and Metabolism of the American Heart Association. Circulation. 2002; 106, 523527.CrossRefGoogle Scholar
Gillman, MW, Rich-Edwards, JW. The fetal origin of adult disease: from sceptic to convert. Paediatr Perinat Epidemiol. 2000; 14, 192193.CrossRefGoogle ScholarPubMed
McMillen, IC, Robinson, JS. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiol Rev. 2005; 85, 571633.CrossRefGoogle ScholarPubMed
McMillen, IC, MacLaughlin, SM, Muhlhausler, BS, Gentili, S, Duffield, JL, Morrison, JL. Developmental origins of adult health and disease: the role of periconceptional and foetal nutrition. Basic Clin Pharmacol Toxicol. 2008; 102, 8289.CrossRefGoogle ScholarPubMed
Morrison, JL. Sheep models of intrauterine growth restriction: fetal adaptations and consequences. Clin Exp Pharmacol Physiol. 2008; 35, 730743.CrossRefGoogle ScholarPubMed
Darby, JRT, Varcoe, TJ, Orgeig, S, Morrison, JL. Cardiorespiratory consequences of intrauterine growth restriction: Influence of timing, severity and duration of hypoxaemia. Theriogenology. 2020; 150, 8495.CrossRefGoogle ScholarPubMed
Barker, DJ, Osmond, C, Golding, J, Kuh, D, Wadsworth, ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. BMJ. 1989; 298, 564567.CrossRefGoogle ScholarPubMed
Barker, DJP. In utero programming of cardiovascular disease. Theriogenology. 2000; 53, 555574.CrossRefGoogle ScholarPubMed
Heindel, JJ, Balbus, J, Birnbaum, L, et al. Developmental origins of health and disease: integrating environmental influences. Endocrinology. 2016; 2016, 1722.Google Scholar
Tamashiro, KL, Terrillion, CE, Hyun, J, Koenig, JI, Moran, TH. Prenatal stress or high-fat diet increases susceptibility to diet-induced obesity in rat offspring. Diabetes. 2009; 58, 11161125.CrossRefGoogle ScholarPubMed
Messer, LC, Boone-Heinonen, J, Mponwane, L, Wallack, L, Thornburg, KL. Developmental programming: priming disease susceptibility for subsequent generations. Curr Epidemiol Rep. 2015; 2, 3751.CrossRefGoogle ScholarPubMed
Thompson, JA, Sarr, O, Piorkowska, K, Gros, R, Regnault, TR. Low birth weight followed by postnatal over-nutrition in the guinea pig exposes a predominant player in the development of vascular dysfunction. J Physiol. 2014; 592, 54295443.CrossRefGoogle ScholarPubMed
Rueda-Clausen, CF, Dolinsky, VW, Morton, JS, Proctor, SD, Dyck, JR, Davidge, ST. Hypoxia-induced intrauterine growth restriction increases the susceptibility of rats to high-fat diet-induced metabolic syndrome. Diabetes. 2011; 60, 507516.CrossRefGoogle ScholarPubMed
Cheong, JN, Wlodek, ME, Moritz, KM, Cuffe, JS. Programming of maternal and offspring disease: impact of growth restriction, fetal sex and transmission across generations. J Physiol. 2016; 594, 47274740.CrossRefGoogle Scholar
Liu, L, Nettleton, JA, Bertoni, AG, Bluemke, DA, Lima, JA, Szklo, M. Dietary pattern, the metabolic syndrome, and left ventricular mass and systolic function: the Multi-Ethnic Study of Atherosclerosis. Am J Clin Nutr. 2009; 90, 362368.CrossRefGoogle ScholarPubMed
Maugeri, A, Hruskova, J, Jakubik, J, et al. How dietary patterns affect left ventricular structure, function and remodelling: evidence from the Kardiovize Brno 2030 study. Sci Rep. 2019; 9, 19154.CrossRefGoogle ScholarPubMed
Dong, F, Ford, SP, Fang, CX, Nijland, MJ, Nathanielsz, PW, Ren, J. Maternal nutrient restriction during early to mid gestation up-regulates cardiac insulin-like growth factor (IGF) receptors associated with enlarged ventricular size in fetal sheep. Growth Horm IGF Res. 2005; 15, 291299.CrossRefGoogle ScholarPubMed
Wang, KC, Tosh, DN, Zhang, S, et al. IGF-2R-Galphaq signaling and cardiac hypertrophy in the low-birth-weight lamb. Am J Physiol Regul Integr Comp Physiol. 2015; 308, R627R635.CrossRefGoogle ScholarPubMed
Morrison, JL, Botting, KJ, Dyer, JL, Williams, SJ, Thornburg, KL, McMillen, IC. Restriction of placental function alters heart development in the sheep fetus. Am J Physiol Regul Integr Comp Physiol. 2007; 293, R306R313.CrossRefGoogle ScholarPubMed
Louey, S, Jonker, SS, Giraud, GD, Thornburg, KL. Placental insufficiency decreases cell cycle activity and terminal maturation in fetal sheep cardiomyocytes. J Physiol. 2007; 580, 639648.CrossRefGoogle ScholarPubMed
Kuo, AH, Li, C, Huber, HF, Schwab, M, Nathanielsz, PW, Clarke, GD. Maternal nutrient restriction during pregnancy and lactation leads to impaired right ventricular function in young adult baboons. J Physiol. 2017; 595, 42454260.CrossRefGoogle ScholarPubMed
Kuo, AH, Li, C, Li, J, Huber, HF, Nathanielsz, PW, Clarke, GD. Cardiac remodelling in a baboon model of intrauterine growth restriction mimics accelerated ageing. J Physiol. 2017; 595, 10931110.CrossRefGoogle Scholar
Darby, JRT, McMillen, IC, Morrison, JL. Maternal undernutrition in late gestation increases IGF2 signalling molecules and collagen deposition in the right ventricle of the fetal sheep heart. J Physiol. 2018; 596, 23452358.CrossRefGoogle ScholarPubMed
Wang, KC, Brooks, DA, Thornburg, KL, Morrison, JL. Activation of IGF-2R stimulates cardiomyocyte hypertrophy in the late gestation sheep fetus. J Physiol. 2012; 590, 54255437.CrossRefGoogle ScholarPubMed
Wang, KC, Zhang, L, McMillen, IC, et al. Fetal growth restriction and the programming of heart growth and cardiac insulin-like growth factor 2 expression in the lamb. J Physiol. 2011; 589, 47094722.CrossRefGoogle ScholarPubMed
Wang, KC, Lim, CH, McMillen, IC, Duffield, JA, Brooks, DA, Morrison, JL. Alteration of cardiac glucose metabolism in association to low birth weight: experimental evidence in lambs with left ventricular hypertrophy. Metabolism 2013; 62, 16621672.CrossRefGoogle ScholarPubMed
Aguila, MB, Mandarim-de-Lacerda, CA. Blood pressure, ventricular volume and number of cardiomyocyte nuclei in rats fed for 12 months on diets differing in fat composition. Mech Ageing Dev. 2001; 122, 7788.CrossRefGoogle ScholarPubMed
Christoffersen, C, Bollano, E, Lindegaard, ML, et al. Cardiac lipid accumulation associated with diastolic dysfunction in obese mice. Endocrinology. 2003; 144, 34833490.CrossRefGoogle ScholarPubMed
Ouwens, DM, Diamant, M, Fodor, M, et al. Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification. Diabetologia. 2007; 50, 19381948.CrossRefGoogle ScholarPubMed
Wilson, CR, Tran, MK, Salazar, KL, Young, ME, Taegtmeyer, H. Western diet, but not high fat diet, causes derangements of fatty acid metabolism and contractile dysfunction in the heart of Wistar rats. Biochem J. 2007; 406, 457467.CrossRefGoogle Scholar
Wadley, GD, McConell, GK, Goodman, CA, Siebel, AL, Westcott, KT, Wlodek, ME. Growth restriction in the rat alters expression of metabolic genes during postnatal cardiac development in a sex-specific manner. Physiol Genomics. 2013; 45, 99105.CrossRefGoogle Scholar
Thompson, LP, Chen, L, Polster, BM, Pinkas, G, Song, H. Prenatal hypoxia impairs cardiac mitochondrial and ventricular function in guinea pig offspring in a sex-related manner. Am J Physiol Regul Integr Comp Physiol. 2018; 315R1232R1241.CrossRefGoogle Scholar
Muralimanoharan, S, Li, C, Nakayasu, ES, et al. Sexual dimorphism in the fetal cardiac response to maternal nutrient restriction. J Mol Cell Cardiol. 2017; 108, 181193.CrossRefGoogle ScholarPubMed
Thompson, LP, Turan, S, Aberdeen, GW. Sex differences and the effects of intrauterine hypoxia on growth and in vivo heart function of fetal guinea pigs. Am J Physiol Regul Integr Comp Physiol. 2020; 319, R243R254.CrossRefGoogle Scholar
Botting, KJ, Loke, XY, Zhang, S, Andersen, JB, Nyengaard, JR, Morrison, JL. IUGR decreases cardiomyocyte endowment and alters cardiac metabolism in a sex- and cause-of-IUGR-specific manner. Am J Physiol Regul Integr Comp Physiol. 2018; 315, R48R67.CrossRefGoogle Scholar
Morrison, JL, Botting, KJ, Darby, JRT, et al. Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic. J Physiol. 2018; 596, 55355569.CrossRefGoogle ScholarPubMed
Grundy, D. Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology. J Physiol. 2015; 593, 25472549.CrossRefGoogle ScholarPubMed
Kilkenny, C, Browne, W, Cuthill, IC, Emerson, M, Altman, DG, Group NCRRGW. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol. 2010; 160, 15771579.CrossRefGoogle ScholarPubMed
Dickinson, H, Moss, TJ, Gatford, KL, et al. A review of fundamental principles for animal models of DOHaD research: an Australian perspective. J Dev Orig Health Dis. 2016; 7, 449472.CrossRefGoogle Scholar
Turner, AJ, Trudinger, BJ. A modification of the uterine artery restriction technique in the guinea pig fetus produces asymmetrical ultrasound growth. Placenta. 2009; 30, 236240.CrossRefGoogle ScholarPubMed
Sarr, O, Thompson, JA, Zhao, L, Lee, TY, Regnault, TR. Low birth weight male guinea pig offspring display increased visceral adiposity in early adulthood. PLoS One. 2014; 9, e98433.CrossRefGoogle ScholarPubMed
Thompson, JA, Gros, R, Richardson, BS, Piorkowska, K, Regnault, TR. Central stiffening in adulthood linked to aberrant aortic remodeling under suboptimal intrauterine conditions. Am J Physiol Regul Integr Comp Physiol. 2011; 301, R1731R1737.CrossRefGoogle ScholarPubMed
Briscoe, TA, Rehn, AE, Dieni, S, et al. Cardiovascular and renal disease in the adolescent guinea pig after chronic placental insufficiency. Am J Obstet Gynecol. 2004; 191, 847855.CrossRefGoogle ScholarPubMed
Kind, KL, Clifton, PM, Grant, PA, et al. Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig. Am J Physiol Regul Integr Comp Physiol. 2003; 284, R140R152.CrossRefGoogle ScholarPubMed
McGillick, EV, Orgeig, S, McMillen, IC, Morrison, JL. The fetal sheep lung does not respond to cortisol infusion during the late canalicular phase of development. Physiol Rep. 2013; 1, e00130.CrossRefGoogle Scholar
Soo, PS, Hiscock, J, Botting, KJ, Roberts, CT, Davey, AK, Morrison, JL. Maternal undernutrition reduces P-glycoprotein in guinea pig placenta and developing brain in late gestation. Reprod Toxicol. 2012; 33, 374381.CrossRefGoogle ScholarPubMed
Bustin, SA, Benes, V, Garson, JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55, 611622.CrossRefGoogle ScholarPubMed
Hellemans, J, Mortier, G, De Paepe, A, Speleman, F, Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007; 8, R19.CrossRefGoogle ScholarPubMed
Lie, S, Hui, M, McMillen, IC, et al. Exposure to rosiglitazone, a PPAR-gamma agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus. Am J Physiol Regul Integr Comp Physiol. 2014; 306, R429R437.CrossRefGoogle ScholarPubMed
Barker, DJ, Winter, PD, Osmond, C, Margetts, B, Simmonds, SJ. Weight in infancy and death from ischaemic heart disease. Lancet. 1989; 2, 577580.CrossRefGoogle ScholarPubMed
Rich-Edwards, JW, Stampfer, MJ, Manson, JE, et al. Birth weight and risk of cardiovascular disease in a cohort of women followed up since 1976. BMJ. 1997; 315, 396400.CrossRefGoogle Scholar
Fall, CH, Osmond, C, Barker, DJ, et al. Fetal and infant growth and cardiovascular risk factors in women. BMJ. 1995; 310, 428432.CrossRefGoogle ScholarPubMed
Fall, CH, Vijayakumar, M, Barker, DJ, Osmond, C, Duggleby, S. Weight in infancy and prevalence of coronary heart disease in adult life. BMJ. 1995; 310, 1719.CrossRefGoogle ScholarPubMed
Frankel, S, Elwood, P, Sweetnam, P, Yarnell, J, Smith, GD. Birthweight, body-mass index in middle age, and incident coronary heart disease. Lancet. 1996; 348, 14781480.CrossRefGoogle ScholarPubMed
Leon, DA, Lithell, HO, Vagero, D, et al. Reduced fetal growth rate and increased risk of death from ischaemic heart disease: cohort study of 15 000 Swedish men and women born 1915–29. BMJ. 1998; 317, 241245.CrossRefGoogle ScholarPubMed
Kajimura, S, Spiegelman, BM, Seale, P. Brown and beige fat: physiological roles beyond heat-generation. Cell Metab. 2015; 22, 546559.CrossRefGoogle Scholar
Taegtmeyer, H, Sen, S, Vela, D. Return to the fetal gene program: a suggested metabolic link to gene expression in the heart. Ann N Y Acad Sci. 2010; 1188, 191198.CrossRefGoogle Scholar
Frey, N, Olson, EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol. 2003; 65, 4579.CrossRefGoogle ScholarPubMed
McMullen, JR, Jennings, GL. Differences between pathological and physiological cardiac hypertrophy: novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol. 2007; 34, 255262.CrossRefGoogle ScholarPubMed
Wei, S, Chow, LT, Shum, IO, Qin, L, Sanderson, JE. Left and right ventricular collagen type I/III ratios and remodeling post-myocardial infarction. J Card Fail. 1999; 5, 117126.CrossRefGoogle ScholarPubMed
Collier, P, Watson, CJ, van Es, MH, et al. Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype. J Mol Cell Cardiol. 2012; 52, 148153.CrossRefGoogle Scholar
Norton, GR, Tsotetsi, J, Trifunovic, B, Hartford, C, Candy, GP, Woodiwiss, AJ. Myocardial stiffness is attributed to alterations in cross-linked collagen rather than total collagen or phenotypes in spontaneously hypertensive rats. Circulation. 1997; 96, 19911998.CrossRefGoogle ScholarPubMed
Kasner, M, Westermann, D, Lopez, B, et al. Diastolic tissue Doppler indexes correlate with the degree of collagen expression and cross-linking in heart failure and normal ejection fraction. J Am Coll Cardiol. 2011; 57, 977985.CrossRefGoogle ScholarPubMed
Owan, TE, Hodge, DO, Herges, RM, Jacobsen, SJ, Roger, VL, Redfield, MM. Trends in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med. 2006; 355, 251259.CrossRefGoogle ScholarPubMed
Cho, WK, Suh, BK. Catch-up growth and catch-up fat in children born small for gestational age. Korean J Pediatr. 2016; 59, 17.CrossRefGoogle ScholarPubMed
Wollmann, HA. Intrauterine growth restriction: definition and etiology. Horm Res. 1998; 49 (Suppl 2), 16.CrossRefGoogle ScholarPubMed
Hales, CN, Barker, DJ. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia. 1992; 35, 595601.CrossRefGoogle ScholarPubMed
Barker, DJ, Martyn, CN, Osmond, C, Hales, CN, Fall, CH. Growth in utero and serum cholesterol concentrations in adult life. BMJ. 1993; 307, 15241527.CrossRefGoogle ScholarPubMed
Christopher, BA, Huang, HM, Berthiaume, JM, et al. Myocardial insulin resistance induced by high fat feeding in heart failure is associated with preserved contractile function. Am J Physiol Heart Circ Physiol. 2010; 299, H1917H1927.CrossRefGoogle ScholarPubMed
McFarlane, SI, Banerji, M, Sowers, JR. Insulin resistance and cardiovascular disease. J Clin Endocrinol Metab. 2001; 86, 713718.Google ScholarPubMed
Park, SY, Cho, YR, Kim, HJ, et al. Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes. 2005; 54, 35303540.CrossRefGoogle ScholarPubMed
Aerni-Flessner, L, Abi-Jaoude, M, Koenig, A, Payne, M, Hruz, PW. GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle. Cardiovasc Diabetol. 2012; 11, 63.CrossRefGoogle ScholarPubMed
Kraegen, EW, Sowden, JA, Halstead, MB, et al. Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4. Biochem J. 1993; 295, 287293.CrossRefGoogle ScholarPubMed
Luiken, JJ, Coort, SL, Koonen, DP, Bonen, A, Glatz, JF. Signalling components involved in contraction-inducible substrate uptake into cardiac myocytes. Proc Nutr Soc. 2004; 63, 251258.CrossRefGoogle ScholarPubMed
Lopaschuk, GD, Collins-Nakai, RL, Itoi, T. Developmental changes in energy substrate use by the heart. Cardiovasc Res. 1992; 26, 11721180.CrossRefGoogle ScholarPubMed
Lopaschuk, GD, Jaswal, JS. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J Cardiovasc Pharmacol. 2010; 56, 130140.CrossRefGoogle ScholarPubMed
Goldberg, IJ, Trent, CM, Schulze, PC. Lipid metabolism and toxicity in the heart. Cell Metab. 2012; 15, 805812.CrossRefGoogle ScholarPubMed
Scarpulla, RC. Nuclear activators and coactivators in mammalian mitochondrial biogenesis. Biochim Biophys Acta. 2002; 1576, 114.CrossRefGoogle ScholarPubMed
Attardi, G, Schatz, G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988; 4, 289333.CrossRefGoogle ScholarPubMed
Park, KS, Kim, SK, Kim, MS, et al. Fetal and early postnatal protein malnutrition cause long-term changes in rat liver and muscle mitochondria. J Nutr. 2003; 133, 30853090.CrossRefGoogle ScholarPubMed
Selak, MA, Storey, BT, Peterside, I, Simmons, RA. Impaired oxidative phosphorylation in skeletal muscle of intrauterine growth-retarded rats. Am J Physiol Endocrinol Metab. 2003; 285, E130E137.CrossRefGoogle ScholarPubMed
Liu, J, Chen, D, Yao, Y, et al. Intrauterine growth retardation increases the susceptibility of pigs to high-fat diet-induced mitochondrial dysfunction in skeletal muscle. Plos One. 2012; 7, e34835.CrossRefGoogle ScholarPubMed
Miranda, S, Foncea, R, Guerrero, J, Leighton, F. Oxidative stress and upregulation of mitochondrial biogenesis genes in mitochondrial DNA-depleted HeLa cells. Biochem Biophys Res Commun. 1999; 258, 4449.CrossRefGoogle ScholarPubMed