C© 2012 Wiley Periodicals, Inc. Birth Defects Research (Part B) 00:1–3 (2012)

Letter to the Editor

Inflammatory Protein Expression in Adolescent andAdult Offspring of Type 1 Diabetic Mice

Daniel Dowling,1,2 Niamh Corrigan,1,2 Paul Downey,3 and Fionnuala M. McAuliffe1,2∗1UCD Obstetrics & Gynaecology, School of Medicine and Medical Science, University College Dublin. National Maternity Hospital,

Dublin, Ireland2UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin. National Maternity Hospital,

Dublin, Ireland3Pathology, National Maternity Hospital, Dublin, Ireland

AIMS: To measure inflammatory markers in offspring of pregestational type 1 diabetic mothers. METHODS: Type 1diabetes was induced in female C57BL6/J mice using streptozotocin. Offspring from control C57BL6/J and type 1 dia-betic mothers were followed up to adulthood and blood was collected at 6 and 12 weeks of age, representing adolescentand adult stages respectively. Five well-established inflammatory markers; Matrix metalloproteinase 9, soluble E-selectin,sICAM-1, sVCAM-1, and total plasminogen activator inhibitor-1 (PAI-1) were measured on an inflammatory multiplexassay in plasma. RESULTS: Blood plasma from adolescent offspring from diabetic mothers displayed an increase in allfive inflammatory markers when compared to controls, and there was a highly significant increase in sVCAM-1 (64.56 ±20.1 vs. 33.8 ± 20.75; p < 0.01) and tPAI-1 (0.05 ± 0.02 vs. 0.02 ± 0.01; p < 0.01) expression. CONCLUSION: Our findingsshow that inflammatory markers are increased in offspring of pregestational diabetic mothers. This may represent a mech-anism for increased risk of cardiovascular disease evident in these offspring. Birth Defects Res (Part B) 00:1–3, 2012. C© 2012Wiley Periodicals, Inc.

Key words: pregestational type 1 diabetes; inflammation; cardiovasculardisease

INTRODUCTIONThe Barker hypothesis states that environmental influ-

ences acting in fetal life are reflected in impaired growthand development that permanently affect structure andmetabolism, leading to increased risk of metabolic diseaselater in life (Barker, 2007). It was Pedersen and Freinkelwho first suggested that maternal fuel metabolism mightexert long-term effects on the future development of theoffspring of diabetic mothers (Pedersen, 1977; Freinkel,1980). Indeed during diabetic pregnancy, the altered nu-trient supply, characterized by maternal hyperglycemiaduring sensitive periods of gestation can lead to an adap-tive response by the fetus, and may contribute to a mod-ification of key metabolic processes and developmentalorganization. Animal and human studies have shownthat maternal diabetes can contribute to the inductionof cardiovascular dysfunction in neonates and adult off-spring (Halliday, 1981; Holemans et al., 1999; Corriganet al., 2009). Though mechanisms are unclear, inflamma-tion is thought to play a critical role in the genesis ofcardiovascular dysfunction. Hypertrophic cardiomyopa-thy is a common pathology affecting up to 75% of fe-tuses in utero and 40% of infants of pregestational diabeticmothers, and can cause clinical symptoms in 5% (Sheehan

et al., 1986; Veille et al., 1992, 1993; Abu-Sulaiman andSubaih, 2004; Russell et al., 2008). Studies have shownthat chronic inflammation can induce myocardial hy-pertrophy, decrease myocardial contractility, and promoteapoptosis and fibrosis, thereby contributing to myocar-dial remodeling (Aukrust et al., 2005). Circulating inflam-matory cytokines have been shown to be elevated in hy-pertrophic cardiomyopathy (Matsumori et al., 1994). Theaim of our study was to measure inflammatory markersin the offspring of pregestational type 1 diabetic mothers,which may explain in part the increased risk of cardio-vascular disease in these offspring. We chose to look atseveral inflammatory markers that have been shown toplay a significant role in the development of cardiovas-cular disease. Plasminogen activator inhibitor-1 (PAI-1) isthe main inhibitor of the fibrinolytic system and elevatedlevels are associated with effects on the development and

∗Correspondence to: Fionnuala M. McAuliffe, UCD Obstetrics & Gy-naecology, School of Medicine and Medical Science, University CollegeDublin, National Maternity Hospital, Dublin 2, Ireland. E-mail: Fionnuala.McAuliffe@ucd.ie

Received 26 July 2012; Accepted 8 August 2012

Published online in Wiley Online Library (wileyonlinelibrary.com/journal/bdrb) DOI: 10.1002/bdrb.21024

2 DOWLING ET AL.

progression of cardiovascular disease (Ploplis, 2011). In-creased PAI-1 levels have also been linked to other riskfactors for vascular disease including hyperinsulinaemiaand insulin resistance (Gough et al., 1993). Vascular adhe-sion molecules including the selectins (E-selectin) and theimmunoglobulin superfamily (ICAM-1 and VCAM-1) areessential for activated immune cells to extravasate fromcirculation into cardiac tissue during acute and chronicinflammation (Malik and Haskard, 1999). Matrix metallo-proteinase 9 (MMP9) has been found to play an importantrole in cardiac remodeling and fibrosis in hypertrophiccardiomyopathy (Roldan et al., 2008; Kitaoka et al., 2011).

METHODSInstitutional ethical approval and Irish government li-

censing were obtained for all procedures. For all ex-periments, the “Principles of laboratory animal care”(NIH publication no. 85–23, revised 1985) were followedthroughout. Type 1 diabetes was induced in femaleC57BL6/J mice using intraperitoneal streptozotocin (STZ)injections. A total of 54 female mice received STZ in-jections, these were divided into six groups and there-fore STZ rounds of injections were given to a group onsix different occasions. The first group (n = 10) received50 mg/kg STZ over five consecutive days; with group2, a dosage regime of 75 mg/kg over 3 days consecu-tively was used. As there was a similar percentage of rateof positive response to the STZ with the later dose, wecontinued with this regime with all other mice as it wasfewer over all injections and therefore less stressful forthe animal, in total 44 females received an STZ dosage of75 mg/kg/3 days. Diabetes was confirmed in femalemice before pregnancy by taking fasting glucose mea-surements over 2 days. Diabetes was diagnosed when afasting glucose was >11 mmol/l on two separate days.Blood was obtained via tail venepuncture and measuredusing an Accu-check compact plus system (Hoffmann-La Roche Ltd. Indianapolis, IN). Female C57BL/6J micewere mated with nondiabetic male C57BL/6J mice. Theday of detection of a vaginal plug was designated em-bryonic day 0.5, then females were placed in an indi-vidual cage for the rest of the pregnancy. Healthy non-diabetic female C57BL6/J mice mated with nondiabeticC57BL/6J males acted as a control group. Mean fast-ing glucose prepregnancy in diabetic mice compared tocontrols was 13.5 ± 1.0 versus 5.4 ± 0.44 mmol/l andat embryonic day 16.5 was 21.7 mmol/l ± 6.3 versus5.3 ± 0.44 mmol/l. For this study, a cohort of animalswas analyzed. Five diabetic mothers delivered 18 pups,12 were sacrificed at 6 weeks and six were sacrificed at12 weeks. While in the control group six control moth-ers delivered 17 pups, 10 were sacrificed at 6 weeks, andseven sacrificed at 12 weeks. Other animals were sacri-ficed for histology examination at various time points.There was no mortality between these time points forother reasons. Blood was taken from the offspring of di-abetic pregnancy at 6 and 12 weeks of age, which repre-sents adolescent and adult developmental stages in mice.Plasma was isolated and analyzed in duplicate on theMilliplex-map mouse cardiovascular disease panel kit.This kit is based on the Luminex xMAP technology (Kanget al., 2012). Microspheres containing a specific capture

antibody for each of the five analytes under investigation(PAI-1 total, sE-selectin, sICAM-1, sVCAM-1, and MMP9)can simultaneously detect and quantify plasma concen-trations of all analytes in each of the 96 wells. Statisti-cal analysis of the data were performed with a statisticalsoftware package (SPSS Win, V.18, SPSS Inc.). Data werenormally distributed and student t-test (two-tailed, equalvariance) was performed to analyze the difference in an-alyte concentration in control and diabetic blood plasma.Data are given as mean ± SD.

RESULTSStatistical analysis of blood plasma obtained from the

offspring of control and type 1 diabetic pregnancy and an-alyzed on the Milliplex-map cardiovascular plate foundthat there were statistically significant differences in someof the inflammatory markers in the adolescent mice(6 weeks of age; Table 1). Concentrations of all five in-flammatory analytes associated with cardiovascular dis-ease were elevated in 6-week adolescent offspring fromtype 1 diabetic pregnancy compared to control mice. Ofthese, soluble VCAM-1 and total PAI-1 displayed signif-icant increases (p < 0.01) in the adolescent offspring ofdiabetic pregnancy. Inflammatory markers in control sub-jects increased from 6 to 12 weeks, whereas in offspringfrom diabetic pregnancy no change was noted from 6 to12 weeks of postnatal age.

DISCUSSIONIn this study, we established a type 1 diabetic preg-

nancy model and investigated whether there was an al-teration in inflammatory protein expression in the adoles-cent and adult offspring. Inflammation has been found toplay a central role in the development and progression ofcardiac remodeling and disease (Matsumori et al., 1994;Aukrust et al., 2005). Our findings show that adolescentoffspring from diabetic pregnancy demonstrate increasedinflammatory protein expression and a highly significantincrease in soluble VCAM-1 and total PAI-1, when com-pared to controls. This is followed by a continued plateauto adulthood. In control offspring, we found a steady in-crease in the expression of inflammatory proteins fromadolescence to adulthood. It is possible that among di-abetic offspring the premature increase in inflammatoryproteins in adolescence initiates the onset of cardiac re-modeling in cardiomyopathy in its various facets, thatis, myocyte disarray, myocardial fibrosis, left ventricularhypertrophy (Elliott and McKenna, 2004) and that thisthen may result in a stabilization of inflammatory pro-tein expression in adulthood. A previous human studyby Manderson et al. ( 2002) found similar results in chil-dren of type 1 diabetic mothers aged 5–11. They found sig-nificant increases in several soluble adhesion moleculesincluding sVCAM-1 and other inflammatory markersincluding PAI-1. Our findings demonstrate that anabnormal intrauterine environment exists in pregesta-tional diabetic pregnancies and this may lead to an al-tered inflammatory state in adolescent offspring. Othershave shown that adverse intrauterine conditions increasethe risk of cardiac disease in the adolescent and this riskcontinues into adulthood in affected offspring (Pedersen,

Birth Defects Research (Part B) 00:1–3, 2012

Letter to the Editor 3

Table 1Mean ± SD of Each Inflammatory Analyte Concentration Quantified from Blood Plasma Obtained from Offspring of

Control and Diabetic Pregnancy at Adolescent (6 Weeks) and Adult (12 Weeks) Stages

6 weeks (Adolescent) 12 weeks (Adult)

Control (n = 12) Diabetic (n = 11) Control (n = 8) Diabetic (n = 6)

MMP9 0.2 ± 0.25 0.37 ± 0.42 0.48 ± 0.57 0.37 ± 0.22sE-selectin 1.64 ± 1.45 1.83 ± 0.65 2.56 ± 1.43 1.43 ± 0.73sICAM-1 0.73 ± 0.26 0.91 ± 0.26 1.06 ± 0.26 0.95 ± 0.14sVCAM-1 33.8 ± 20.75 64.56 ± 20.1** 60.61 ± 21.3 52.04 ± 15.93tPAI-1 0.02 ± 0.01 0.05 ± 0.02** 0.03 ± 0.013 0.02 ± 0.01

Two-tailed student t-test, equal variance assumed < 0.01**. Asterisk indicates level of significance.

1977; Freinkel, 1980; Holemans et al., 1999; Mandersonet al., 2002; Barker, 2007; Dowling and McAuliffe, 2012).

CONCLUSIONOur findings show that inflammatory markers are in-

creased in offspring of pregestational diabetic mothers.This may represent a mechanism for cardiovascular dam-age in these offspring.

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