Progeria Syndrome

Mechanisms of Premature Vascular Aging in Children WithHutchinson-Gilford Progeria Syndrome

Marie Gerhard-Herman, Leslie B. Smoot, Nicole Wake, Mark W. Kieran, Monica E. Kleinman,David T. Miller, Armin Schwartzman, Anita Giobbie-Hurder, Donna Neuberg, Leslie B. Gordon

Abstract—Hutchinson-Gilford progeria syndrome is a rare, segmental premature aging syndrome of acceleratedatherosclerosis and early death from myocardial infarction or stroke. This study sought to establish comprehensivecharacterization of the fatal vasculopathy in Hutchinson-Gilford progeria syndrome and its relevance to normal aging.We performed cardiovascular assessments at a single clinical site on the largest prospectively studied cohort to date.Carotid-femoral pulse wave velocity was dramatically elevated (mean: 13.00�3.83 m/s). Carotid duplex ultrasoundechobrightness, assessed in predefined tissue sites as a measure of arterial wall density, was significantly greater thanage- and sex-matched controls in the intima-media (P�0.02), near adventitia (P�0.003), and deep adventitia (P�0.01),as was internal carotid artery mean flow velocity (P�0.0001). Ankle-brachial indices were abnormal in 78% of patients.Effective disease treatments may be heralded by normalizing trends of these noninvasive cardiovascular measures. Thedata demonstrate that, along with peripheral vascular occlusive disease, accelerated vascular stiffening is an early andpervasive mechanism of vascular disease in Hutchinson-Gilford progeria syndrome. There is considerable overlap withcardiovascular changes of normal aging, which reinforces the view that defining mechanisms of cardiovascular diseasein Hutchinson-Gilford progeria syndrome provides a unique opportunity to isolate a subset of factors influencingcardiovascular disease in the general aging population. (Hypertension. 2012;59:92-97.) ● Online Data Supplement

Key Words: Hutchinson-Gilford progeria syndrome � atherosclerosis � arteriosclerosis � arterial stiffness � lamin � aging

Hutchinson-Gilford progeria syndrome (HGPS) is anextremely rare (incidence: 1 in 4–8 million) sporadic

genetic disorder involving aberrant splicing of the LMNAgene,1,2 resulting in the production of a disease-causingmutant lamin A protein called progerin.1 Lamin A3 and, thus,progerin,4 are inner nuclear membrane proteins expressed inall layers of the vasculature in both HGPS and, at a reducedrate, in normal aging.4 Select clinical features of this disordermimic those of accelerated human aging, with prematurecardiovascular disease (CVD) as the basis for significantmorbidity and mortality.5 Individuals with HGPS die at anaverage age of 13 years because of myocardial infarction orstroke.6 The rapid progression of CVD in HGPS presents anopportunity to explore the natural history of human CVDevolving under the influence of progerin and in relativeisolation from the influences of diet, smoking, family history,and hypercholesterolemia.

Robust characterization of CVD in HGPS is essential fordeveloping clinical standards of care, for deriving objectivecardiovascular end points when assessing treatment efficacy,and for exploring the intersections between HGPS and the

general aging population. To date these issues have beenmodestly characterized.7 Based on dense arterial proteogly-can and collagen deposition in HGPS human4,8 and mousemodel9 autopsies, along with abnormally echobright vascula-ture on carotid ultrasound in our previous natural historystudy,5 we hypothesized that end-stage cardiovascular eventsin HGPS are related to progressive impairment in vascularcompliance. We, therefore, conducted a single center clinicaltrial involving the largest cohort of subjects with HGPS todate, using measures known to be associated with vascularstiffening in addition to established cardiovascular risk factorsthat influence CVD through independent mechanisms. Here weexamined variables with potential clinical use in HGPS asmeasures of both disease severity and treatment efficacy for thisunique model of accelerated cardiovascular aging.

MethodsStudy Population and DesignTwenty-six children with classic p.G608G HGPS (c.1824 C�T inLMNA) were enrolled into a clinical trial for the study of progeria(NCT00425607). Several children were unable to perform sometests, and in those cases, the number was �26. Sixty-two age- and

Received August 10, 2011; first decision September 6, 2011; revision accepted October 24, 2011.From the Division of Cardiology (M.G.-H., N.W.), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; Departments of

Cardiology (L.B.S.), Anesthesia (M.E.K., L.B.G.), Genetics (D.T.M.), and Pediatric Oncology (M.W.K.), Children’s Hospital Boston and HarvardMedical School, Boston, MA; Division of Pediatric Oncology (M.W.K.) and Department of Biostatistics and Computational Biology (A.S., A.G.-H.,D.N.), Dana-Farber Cancer Institute, Boston, MA; Department of Biostatistics (A.S., D.N.), Harvard School of Public Health, Boston, MA; Departmentof Pediatrics (L.B.G.), Hasbro Children’s Hospital, Warren Alpert Medical School of Brown University, Providence, RI.

Correspondence to Leslie B. Gordon, Department of Pediatrics, Hasbro Children’s Hospital, 593 Eddy St, Providence, RI 02903. E-mail Leslie.Gordon@brown.edu© 2011 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.111.180919

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sex-matched healthy control children without history of bloodpressure (BP) or cardiac abnormalities were enrolled to establishnormative pediatric reference data for parameters not available in theliterature. These were internal carotid artery (ICA) echodensity andflow velocity, both obtained from a single carotid ultrasound.Fifty-seven subjects provided useful data for analysis. The Chil-dren’s Hospital Boston Committee on Clinical Investigation ap-proved the study protocol.

Written informed consent was obtained from parents and studyassent from children aged �7 years. Participants with HGPS from 16countries were flown to Children’s Hospital Boston for evaluations.Consent was provided in written and oral form in the language of theparents; interpreters were provided during testing periods for non-English–speaking participants. Outside medical charts and clinicalinformation were obtained either from the Progeria Research Foun-dation Medical and Research Database (principal investigator,L.B.G.), with parental consent (Brown University Center for Geron-tology and Healthcare Research, Providence, RI), or directly fromparents and referring physicians.

Clinical MeasuresA complete history and physical, venous blood collection, oralglucose tolerance testing, 12-lead ECG, and automated BP measureswere performed. Either a pediatric 12- to 19-cm or infant size 8- to13-cm BP cuff was selected for each child, based on the size thatwould allow the bladder to cover 80% of the upper arm. Insulinresistance was determined using the following equation: homeostasismodel assessment-insulin resistance�(fasting glucose (mg/dl)) �(fasting insulin (�U/mL))/405. Height-age was determined by cal-culating the median age in the general population of a child with theheight of each patient with HGPS, using Centers for Disease Controland Prevention sex-specific pediatric growth curves (http://www.cdc.gov/growthcharts/). All of the other studies were performed by asingle cardiologist (M.G.-H.) in the morning, with the subjects fastingand resting supine in a quiet darkened, temperature-controlled (22°C)room.

Diagnostic carotid artery ultrasonography was performed in anIntersocietal Commission for the Accreditation of Vascular Laborato-ries–accredited laboratory using established protocols. A Philips iU22ultrasound machine (Philips, Eindhoven, the Netherlands) equippedwith a 17- to 5-MHz broadband linear-array transducer was used.

Velocity was obtained using pulsed wave Doppler performed withappropriate angle correction. Because the left-sided vessels wereroutinely performed later in the examination when the children mighthave been more tired or agitated, we assessed right and left sidesseparately to assure that any minor changes in vessel physiologywould be detected. Mean distal ICA velocity was derived using onethird of the peak systolic velocity plus two thirds of the end diastolicvelocity, as described previously.10 Gray map 5 was used uniformlyafter adjusting overall gain so that intraluminal blood appearedblack. Digital gain compensation was kept perpendicular.

Distal common carotid artery far-wall intima-media thickness(IMT) was measured from the intima-lumen border to the media-adventitia border over a 2-cm segment according to standardprotocol using edge-detection software (Medical Imaging Applica-tions, Coralville, IA).11 Carotid-femoral pulse wave velocity(PWVcf) and ankle-brachial index (ABI) measurements were deter-mined as described previously.12,13 For detailed protocols, please seethe online Data Supplement at http://hyper.ahajournals.org.

Echodensity values were quantified using ImageJ software (Na-tional Institutes of Health, Bethesda, MD) on a grayscale rangingfrom 0 (black) to 256 (white), where 0 was calibrated to equal thedensity of intraluminal blood (preset to appear as black).14 Prespeci-fied rectangular regions were 1 cm in length, starting 1 cm proximalto the carotid bifurcation within the far wall of the right distalcommon carotid artery. Depths for prespecified sampling areas werethe intima-media (from the intima-lumen interface to the media-adventitia border), the near adventitia (from the media-adventitiaborder to 0.4 mm depth), and the deep adventitia (from 1.6 to 2.0 mmdepth beneath the media/adventitia interface). Histograms and per-

centiles were calculated using MATLAB 7.9 (The Mathworks, Inc,Natick, MA).

Statistical AnalysesStatistical comparisons between sex, age groups, or cases/controlswere conducted using Wilcoxon rank-sum tests to account forasymmetry of distribution and nonhomogeneity of variance. Spear-man correlation coefficients were used to quantify relationshipsbetween continuous variables. Statistical significance was defined asP�0.05; there were no corrections for multiple comparisons.

Results

Demographic and Anthropometric ProfilesAnthropometric and historical cardiovascular characteristicsare presented in Table S1, available in the online DataSupplement. Mean age was 7.4�3.4 years (range: 3.1–16.2).All of the patients fell well below the third percentile forheight (94.96�11.6 cm), weight (10.46�2.7 kg), and bodymass index (11.5�1.2 kg/m2). Mean height-age was 3.4�1.5years, which is 4.1 years younger than chronologic age.

Metabolic ProfileMetabolic profile data are presented in Table S2. With oralglucose tolerance testing, only a small fraction of patients hadabnormal fasting (16%) or 2-hour glucose levels (8%). Incontrast, 52% of patients had hyperinsulinemia, and 36%were insulin resistant. One patient with insulin resistance hadaccompanying glucose intolerance, and 1 patient, age 16years, had diabetes mellitus.

Serum lipid profiles agreed with previous studies (TableS2).5,15 High-density lipoprotein was significantly low in 8(33%) of 24 patients. Mean cholesterol, low-density lipoprotein,and triglycerides were in the normal range. Leptin was unde-tectable or below normal in 92% of patents. All of the patientshad normal liver and kidney function, assessed with serumaspartate aminotransferase, alanine aminotransferase, bilirubin(total and direct), serum urea nitrogen, and creatinine.

ECG FindingsEighty-five percent of patients (22 of 26) had normal 12-leadECGs. Four patients (15%; ages 9–16 years) had significantECG abnormalities: 2 exhibited borderline left ventricularhypertrophy (1 with atrial enlargement), and 2 exhibited leftventricular hypertrophy with strain.

Blood PressureWe evaluated BP in relationship to both chronologic age andheight-age. When assessed by chronologic age, systolic anddiastolic pressures were �95th percentile in 7 (27%) of 26and 9 (35%) of 26 patients, respectively. Using height-agenorms to account for patient size, systolic and diastolic pressureswere elevated in 12 (46%) of 26 and 14 (54%) of 26 patients,respectively. Four of 26 children were taking antihypertensivemedications (3 taking �-blockers and 1 taking an angiotensin-converting enzyme inhibitor). Elevations in both systolic anddiastolic BPs resulted in overall pulse pressure within the normalrange for both chronologic age and height-age (n�25; median:41 mm Hg; range: 24–65 mm Hg).

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Carotid-Femoral Pulse Wave VelocityMean PWVcf was 13.00�3.83 m/s (n�21), which is 228% ofthe highest published mean pediatric normal values of5.7�0.9 m/s.16 (Figure 1), and mean values were comparableto those of typical adults �60 years of age.17,18 The Spearmancorrelation coefficient between PWVcf and homeostasismodel assessment-insulin resistance was 0.456 (P�0.04).PWV was significantly increased (P�0.03) in the childrenwho were insulin resistant (n�8; PWVcf median: 16.1; range:10.1–18.8) versus those who were not (n�12; PWVcf me-dian: 12.6; range: 7.2–17.5).

Diagnostic Carotid UltrasonographyAtherosclerotic plaque limited to the ICA was evident in 2(8%) of 26 patients (Figure S1). One subject had totalocclusion of the ICA. No other significant stenoses wereevident by systolic velocity ratios.

Mean distal ICA flow velocities10 were assessed as ameasure of distal small vessel function. We observed in-creased flow velocities bilaterally in �80% of the HGPScohort compared with controls (P�0.0001 for both right- andleft-sided comparisons; Figure 2). To assess flow abnormalityby patient, we next calculated the fraction of children withHGPS whose mean velocities were �95th percentile of

children in the control cohort. With right and left ICA flowvelocities averaged, 76.0 cm/s represented the 95th percentilein the control group; values above this were defined aselevated. In the HGPS population, 15 children (63%) hadbilateral flow velocity elevation; 4 children (17%) had uni-lateral flow velocity elevation; and 5 children (21%) werewithin normal limits bilaterally.

Intima-Media ThicknessSimilar to our previous study,5 all of the 26 children withHGPS had IMT measures using right, left, and combinedICA values (each 0.43�0.03 mm) within the normal rangeand similar to controls for right (n�55; 0.40�0.04 mm),left (n�54; 0.39�0.04 mm), and combined values (0.40�0.03 mm).19

Common Carotid Artery EchodensityVascular echobrightness on ultrasound increases with tissuedensity.20,21 Carotid artery wall echodensity was assessedwithin predefined areas of the intima-media, near adventitia,and deep adventitia in 22 HGPS subjects and compared with52 age- and sex-matched controls (Figure 3A through 3D).Predefined areas were transformed into histograms represent-ing the spectrum of echobrightness for the entire area cap-tured (Figure 3E through 3G). Most control histograms(Figure 3E) and some HGPS histograms (Figure 3F) werefully captured on the grayscale. However, in some cases, thevessel wall was uniformly white beyond the limit of echode-tection, resulting in a histogram compressed against the upperlimit wall (Figure 3G). To better define the differences withthese data, we compared density at both the 10th and 50thpercentiles for each subject. At both the 10th and 50th percen-tiles, mean wall echodensities of the intima (10th percentileP�0.0001; 50th percentile P�0.02), near adventitia (10th per-centile P�0.02; 50th percentile P�0.003), and deep adventitia(10th percentile P�0.03; 50th percentile P�0.01) were signifi-cantly greater in the HGPS cohort when compared with controls(Figure 3H). There was no significant association betweenPWVcf and echodensity at the 10th and 50th percentiles withineach of the echodensity sites (n�19).

Ankle-Brachial IndicesPatients exhibited both elevated and depressed ABI (Table).Seventy-eight percent of children had ABI abnormality in oneor both limbs; only 5 children (22%) were within normallimits bilaterally, with ABI values between 1.0 and 1.2.

DiscussionAccelerated CVD leads to debilitating morbidity in HGPSand culminates in mortality from myocardial infarction orstroke at an average at of 13 years.6 For the first time, we haveidentified elevated PWVcf, increased intima-media and ad-ventitia echodensity, abnormal ABI, and increased ICA meanflow velocity as pervasive disease features in HGPS. Evi-dence of vascular dysfunction is apparent in 100% of ourcohort, in children as young as 3 years of age. ElevatedPWVcf and wall echodensity identify vascular stiffening as anearly and important contributor to cardiovascular decline;abnormalities in ABI and distal ICA mean flow velocity

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Figure 1. Carotid-femoral pulse wave velocity (PWVcf) is ele-vated in Hutchinson-Gilford progeria syndrome (HGPS); PWVcffor each of 21 children with HGPS. Solid horizontal bar repre-sentes published pediatric normal range (mean�SD).16

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underscore the presence of arterial occlusive disease. Takentogether, these abnormalities promote dynamic increases inventricular afterload, impaired vascular autoregulation, andcardiac and cerebrovascular decompensation.

We identified 3 main contributors to vascular stiffening.First, PWVcf was dramatically elevated overall. This findingis consistent with the elevated augmentation index detectedpreviously in HGPS.5 PWV is inversely related to arterialwall distensibility and serves as the gold standard for mea-sures of arterial stiffness.22 Moreover, increased PWV ispredictive of subsequent cardiovascular events. Second, we

discovered abnormally echodense vascular walls in the HGPScohort. We hypothesize that this corresponds with the dra-matically thickened, fibrotic matrix detected previously onhistopathologic analyses of both children with HGPS4,8 and atransgenic mouse model of HGPS.9 These autopsy studieshave described a diffuse arteriopathy marked by strikingdepletion of vascular smooth muscle cells from the media,along with deposition of replacement proteoglycans andcollagen in both media and adventitia. Such maladaptivevascular remodeling would predict lower arterial complianceand impaired flow reserve. Third, in agreement with ourprevious study,5 we found that a significant fraction ofpatients had systolic and diastolic BPs above the 95thpercentile. This fraction increased after adjusting for height-age, because the HGPS cohort is extremely small for age, andsomatic size and chronologic age contribute to BP.23 Theseelevated BP trends would be expected in the setting ofsignificant vascular stiffness, because prehypertensive adultsdisplay both impaired arterial functions and elevated PWV.24

The observed elevation in PWVcf without elevation of pulsepressure in this cohort may be related to overestimation ofdiastolic pressure by the automated cuff, altered volumestatus and heart rate in fasting patients, or disproportionatechanges in the aorta compared with the peripheral arteries.Characteristics of large versus small vessel disease and theirrelative contribution to pulse pressure and systemic vascular

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Figure 3. Increased carotid artery echodensity in Hutchinson-Gilford progeria syndrome (HGPS). A, Longitudinal image of the commoncarotid artery (CCA). L indicates lumen. Area of interest indicated by large dashed rectangle. B, Enlargement of the area of interest seenin A, showing echodensity assessed in prespecified areas indicated by the dashed boxes in the distal common carotid artery (CCA) far wall.White dashed box indicates intima-media area, and black dashed boxes indicated superficial and deep adventitial areas of measurement. C,Posterior CCA wall in control subject demonstrating normal echodensity of the intima (I), media (M), and adventitia (A), compared with D,increased echodensity noted in the HGPS posterior CCA wall. E through G, Sample histogram plots showing pixel intensity (x axis) vs pixelcount (y axis) derived from the deep adventitia site in E, a control child, and F and G, children with HGPS. Solid vertical line represents 50thpercentile and dashed vertical line represents 10th percentile for each plot. H, Mean (�SE) values for intima-media density, superficial adven-titia density, and deep adventitia density at the 10th percentile in the control (white bar; n�53) and HGPS (black bar; n�23) groups. P valuesfor HGPS vs controls were *P�0.05 and **P�0.001.

Table. Blood Pressure and Ankle-Brachial Index

Blood Pressure (n�26) Systolic Diastolic

Median (range), mm Hg 104 (80–134) 69.5 (40–96)

�95 percentile for chronologic age, n (%) 7 (27) 9 (35)

�95 percentile for height-age, n (%) 12 (46) 14 (54)

ABI Stratification (n�23)No. of Subjects With1 Limb in Category

No. of Subjects With2 Limbs in Category

High abnormal, �1.20 3 2

Normal range, 1.01–1.20 9 5

Borderline low, 0.91–1.00 7 4

Low abnormal, 0.80–0.90 5 0

ABI indicates ankle-brachial index.

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resistance will be important in understanding the basis for themarked abnormalities observed in PWVcf. Future HGPSstudies will benefit from rigorous longitudinal assessment ofthese parameters in both static and dynamic states.

We experienced several study limitations. In young chil-dren, vascular testing is limited by the child’s ability towithstand testing and by a paucity of standards for compar-ison. Where pediatric standards were unavailable to quanti-tatively evaluate vessel wall density and distal ICA meanflow velocity, we tested an age- and sex-matched healthycontrol cohort, with predictably larger body mass index.Where possible, we relied on comparison with publishedpediatric normal control studies (eg, PWVcf), which lendsitself to confounding factors, such as interrater reliability. Inaddition, noninvasive carotid artery ultrasonography is sub-ject to technical limitations. Although we used a pediatricultrasound transducer with the highest available frequency,the relative lack of subcutaneous neck tissue5 precludedadequate image resolution along the carotid arterial near wallin many cases. Therefore, all of the measures were acquiredfrom the far wall according to standard protocol11 and whereimage resolution was excellent for all of the subjects.Echodensity in the HGPS cohort often saturated the upperlimit of detection; technical expansion of the 256 grayscalewould allow additional density capture. Finally, longitudinalanalyses may further clarify the relationship of age to thevasculopathy of HGPS in future studies.

Understanding the intersections and distinctions betweenHGPS and normal aging can inform both fields of study and helpus to interpret the clinical influence that progerin may have aftera lifetime of low level accumulation in aging4,25,26 versus itsintense production in children with HGPS. Given the mountingbody of published studies that demonstrate progerin’s presencein aging vasculature, as well as its link to telomere dysfunctionin cellular senescence,27 it is likely that the disease manifested inHGPS is an intensified representation of a subset of the factorsthat influence CVD in normal aging. Between the two, we seesome strong overlap, weaker associations, and an echodensitymeasure that is as yet undefined in aging.

PWV, elevated BP, and insulin resistance paint a picturesimilar to that of the usual aging process. In particular, the HGPScohort exhibited mean PWVs of 13.00�3.83 comparable tothose of adults over age 60 years.17,18 Redheuil et al18 found thatPWVcf increased from 6.2�0.7 m/s at ages 20 to 29 years tovalues comparable to our HGPS population at ages 60 to 69years (12.8�3.9 m/s) and over 70 years (13.8�5.3 m/s) andexhibited a highly significant relationship with aging. Althoughinsulin resistance and increased BP may have some contributionto increased PWV in our HGPS cohort, as they do in non-HGPSpatients with diabetes mellitus and hypertension,28–30 onlychronologic aging is independently associated with the degree ofPWV elevations that we detect in HGPS.

The end-stage manifestation of atherosclerotic disease, thepresence of carotid plaque, was found in only 2 of our olderHGPS subjects. As with aging, this implies that visible plaqueoccurs later in disease. In contrast, elevated mean ICA flowvelocity was seen across all ages in HGPS. In aging,increased ICA flow velocity directly correlates with degree ofstenosis.31 This abnormality may be an early indicator of an

ongoing process that culminates in plaque formation, as aconsequence of narrowing of the distal ICA. Alternatively,elevated ICA flow velocity may be attributed to increasedflow within the brain because of collateral formation aroundocclusive disease.

ABI is a useful tool to detect peripheral vascular disease inthe general aging population, where it indicates calcificationsor vessel stiffness when high, as well as lower extremityocclusive disease when low.12 Both high and low ABI arepredictors for cardiovascular mortality. We detected bothelevated and depressed ABI in HGPS, which likely reflects acomposite picture of peripheral vascular stiffening and occlu-sive disease that could actually yield misleading normal ABIvalues in this mixed disease setting.

Echodensity is an emerging area of study in cardiology andaging. Echobrightness on ultrasound increases as tissue densityincreases; each pixel represents one returning echo at a particulardepth where the ultrasound encounters tissue. The brightness ofan image is determined by the number of returning echos, whichis proportional to the density of the tissue being evaluated.21 Wefound that the intima-media complex in HGPS is of normalthickness but abnormal echodensity. This relationship betweenechogenicity and tissue pathology can occur in the face ofnormal IMT. Lind et al14 performed a study of human agingcommon carotid artery vasculature in randomly chosen peopleover age 70 years. Those with normal IMT had the sameincreased echogenicity as those with widened IMT within boththe intima-media and within plaques. In diseased cardiovasculartissue and plaque, both animal and human studies have sup-ported duplex ultrasound as a useful tool for detecting tissuepathology with dysregulated extracellular matrix through in-creased echogenicity. When correlating echogenicity with tissuepathology in rats, Tabel et al20 demonstrated hyperechoic tissueonly within older infarcts where thick collagen fibers werepresent. New infarcts with thinner, normal-looking collagenfibrils were not hyperechoic. This dense fibrous matrix isreminiscent of the vascular pathology seen in HGPS.4,8 Al-though arterial intima-media density has been correlated withcardiovascular risk factors and carotid plaque echogenicity inelderly adults,14 the striking degree of adventitial abnormalitiesobserved in this study has not yet been evaluated in aging andshould be the subject of future studies.

PerspectivesThe HGPS vascular phenotype is associated with significantpremature cardiovascular morbidity and mortality occurringin the absence of the common comorbidities that are preva-lent in adult populations, such as smoking, hypercholesterol-emia, and obesity. We characterized this single gene disorderas a disease of vascular stiffening in the setting of gradualvascular stenosis, much like that seen over a lifetime innormative aging. This study’s human clinical investigationshelp us to translate the in vitro,7,9,26,27,32 mouse model,9,33 andhuman pathological4,25 support for key roles of altered laminA and progerin in human aging. In addition, noninvasivemeasures, including PWVcf, carotid wall echodensity andICA flow velocity, offer quantitative insights into acceleratedvasculopathy in HGPS and may provide sensitive indicatorsof disease progression or remission with adjuvant therapies.

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AcknowledgmentsWe are grateful to the children with progeria and their families andto the children who participated as control subjects. We thank KellyLittlefield, Kiera McKendrick, and Susan Campbell for data abstrac-tion and management and for patient coordination and Susan Chengfor assistance with carotid artery thickness and densitymeasurements.

Sources of FundingThe study was funded by the Progeria Research Foundation(PRFCLIN2007-01), Peabody, MA and by National Institutes ofHealth grants to the Children’s Hospital Boston General ClinicalResearch Center (MO1-RR02172) and to the Harvard CatalystClinical and Translational Study Unit (UL1 RR025758-01).

DisclosuresL.B.G. is the parent of a child with HGPS who participated in thisstudy.

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GordonDavid T. Miller, Armin Schwartzman, Anita Giobbie-Hurder, Donna Neuberg and Leslie B.

Marie Gerhard-Herman, Leslie B. Smoot, Nicole Wake, Mark W. Kieran, Monica E. Kleinman,Syndrome

Mechanisms of Premature Vascular Aging in Children With Hutchinson-Gilford Progeria

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2011 American Heart Association, Inc. All rights reserved.

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1

ONLINE SUPPLEMENT

MECHANISMS OF PREMATURE VASCULAR AGING IN CHILDREN WITH

HUTCHINSON-GILFORD PROGERIA SYNDROME

Marie Gerhard-Herman, Leslie B. Smoot, Nicole Wake, Mark W. Kieran, Monica E. Kleinman,

David T. Miller, Armin Schwartzman, Anita Giobbie-Hurder, Donna Neuberg, Leslie B. Gordon

From the Division of Cardiology (M.G-H.), Brigham and Women's Hospital and Harvard

Medical School, Boston, MA, 02115; Departments of Cardiology (L.B.S.), Anesthesia (M.E.K,

L.B.G.), and Genetics (D.T.M.), and Pediatric Oncology (M.W.K.), Children’s Hospital Boston

and Harvard Medical School, Boston, MA 02115; Division of Pediatric Oncology (M.W.K.) and

Department of Biostatistics and Computational Biology (A.S., A.G-H, D.N.), Dana-Farber

Cancer Institute, Boston, MA 02115; Department of Biostatistics (A.S., D.N.), Harvard School

of Public Health, Boston, MA; Department of Pediatrics (L.B.G.), Hasbro Children’s Hospital,

Warren Alpert Medical School of Brown University, Providence, RI 02912

Short Title: Vascular Aging in Progeria

Correspondence to: Leslie B. Gordon, MD, PhD

Department of Pediatrics, Hasbro Children's Hospital, 593 Eddy Street, Providence, RI, 02903

Phone: 978-535-2594 ; Fax: 508-543-0377 ; Email: Leslie.Gordon@brown.edu

2

Online-only Methods: Carotid-femoral pulse-wave velocity (PWVcf) measurements were determined by using a

duplex ultrasound with simultaneous ECG acquisition to measure the propagation time of the pressure pulse from the carotid to femoral arteries. The onset of the arterial pulse waveform was identified by using the intersecting tangent method.1 Propagation time (∆tcf) was calculated by measuring the time lag between the R-wave of the simultaneous ECG and the arrival of the arterial pulse at both the carotid (∆tc) and (∆tf) femoral arteries. The distance between the carotid and femoral arteries (lcf) was measured and recorded. PWVcf was calculated using the formula PWVcf = lcf/∆tcf. Published normal ranges used for comparison were performed using this method.2, 3

Ankle brachial indices (ABIs) were performed using appropriate sized pediatric

sphygmomanometers with subjects in the supine position after resting for ten minutes. Systolic pressures were obtained in each arm and ankle. A Doppler probe and photoplethysmograph were used to monitor the pulse while the sphygmomanometer was inflated to suprasystolic pressure above each artery. The sphygmomanometer was then deflated and the pressure at which the pulse returned was recorded. ABIs were calculated by dividing the systolic ankle pressure by the highest systolic arm pressure.

References:

1. Determinants of pulse wave velocity in healthy people and in the presence of cardiovascular risk factors: 'Establishing normal and reference values'. European Heart Journal. 2010;31:2338-2350.

2. Sakuragi S, Abhayaratna K, Gravenmaker KJ, O'Reilly C, Srikusalanukul W, Budge MM, Telford RD, Abhayaratna WP. Influence of adiposity and physical activity on arterial stiffness in healthy children: The lifestyle of our kids study. Hypertension. 2009;53:611-616.

3. Simonetti GD, Eisenberger U, Bergmann IP, Frey FJ, Mohaupt MG. Pulse contour analysis: A valid assessment of central arterial stiffness in children? Pediatr Nephrol. 2008;23:439-444.

4. Gordon C, Gordon L, Snyder B, Nazarian A, Quinn N, Huh S, Giobbie-Hurder A, Neuberg D, Cleveland R, Kleinman M, Miller D, Kieran M. Hutchinson-gilford progeria syndrome represents a unique skeletal dysplasia. J Bone and Mineral Res. 2011;26:1670-1679.

3

Online-only Results:

Table S1. HGPS Cohort Anthropometrics and CV History (n=26*)

Anthropometrics at Study Entry† Mean ±SD Chronologic Age 7.4 ± 3.4 Height Age 3.4 ± 1.5 Weight (kg) 10.46 ± 2.7 Standing Height (cm) 94.96 ± 11.6 BMI (kg/m2) 11.5 ± 1.2

Historical CV Characteristics N %

Type of CV events Cerebrovascular accident 4 15% Supraventricular tachycardia 2 8% Congenital heart disease 1 4%

ROSS Classification I-asymptomatic 23 88% II-exertional symptoms 3 12%

History of Hypertension 5 19% History of Treatment for Hypertension

ACE-I or β-blocker 4 22% Diuretic 2 8%

*11 males (42%) and 15 females (58%); all tanner stage 1 † anthropometrics are also included in Gordon et al.4

4

Table S2. Glucose Metabolism and Lipid Values in HGPS Cohort

Serum Test Mean ±SD Fraction (%) of Patients in Abnormal Range* Normal Range

Fasting Glucose (mg/dl) 88.32±13.73 2/25 (8%) <100

OGTT 2 hr Glucose (mg/dl) 106.1±33.9 3/25 IGT (12%)

1/25 Diabetic (4%)

Normal <140 Impaired glucose tolerance 140-200 Diabetes >200

Fasting Insulin (uU/mL) 22.98±42.41 13/25 (52%) <5

†HOMA - IR 5.6+/-10.3 9/25 (36%) Normal <2.5

Leptin (µg/L) not assessable

22/26 Undetectable (84%) 2/26 Below Normal (8%)

Girls 1.23-5.12 Boys 0.65-3.04

HbA1C (%) 5.58±0.55 1/25 (4%) <6

‡Total Cholesterol (mg/dl) 149.28±30.03 1/25 Borderline (4%)

2/25 High (8%)

Desirable:<170 Borderline 170-199; High≥>200

HDL cholesterol (mg/dl) 43.67±14.38 8/24 (33%) Desirable: >35

LDL cholesterol (mg/dl) 85.52±31.83 2/23 Borderline (9%)

1/23 High (4%)

Desirable: <110 Borderline: 110-129 High:≥ 130

Triglycerides (mg/dl) 91.19±93.35 1/25 Borderline (4%)

2/25 High (8%)

Desirable: <150 Borderline 150-199; High≥200

*In cases where patients were unable to fast, results are reported for fewer than 26 patients † homeostasis model assessment-insulin resistance (HOMA-IR) = fasting (glucose)(insulin)/405 ‡Five patients were taking cholesterol-lowering drugs (4 taking statins and one taking ezetimibe) at the time of study visit

5

L P

B

A

No Flow Positive Flow

Figure S1. Carotid occlusions. Carotid ultrasonography in two HGPS individuals demonstrating A, complete distal internal carotid stenosis. Note colored area of Doppler flow (Positive Flow) prior to total occlusion (No Flow). B, a non-occlusive atherosclerotic plaque (P) juts out within the vascular lumen (L).