Cardiovascular disease (CVD) and stroke account for the majority of adult deaths in the industrialized world, and are now major causes of morbidity and mortality in the developing world [7]. In the U.S., atherosclerosis is the main cause of CVD-related heart attacks and strokes [8]. PAD, affecting limb circulation in 10 million Americans, is primarily caused by atherosclerosis and is a risk indicator for coronary and carotid arterial disease, aneurysm, diabetes, and hypertension [9]. Diagnosis of PAD increases risk of CVD mortality 4–6 times over healthy age-matched individuals, though PAD is often asymptomatic [6]. The prevalence of PAD is 10–25% in men and women over 55 in developed countries.

Recent research considers chronic inflammation in the onset and progression of CVD. Many studies associate inflammatory markers and CVD morbidity and mortality after controlling for risk factors [8], [10], [11]. Bacterial, viral and parasitic infections are common among traditional human and primate populations both now and throughout our evolutionary past [12]. As a consequence, inflammation and adaptive immune responses have evolved under intense selection against pathogens. However, in modern societies with immunizations, public sanitation, adequate nutrition and medical services, and where hypertensive people live sedentary lives and frequently smoke, inflammatory processes have been implicated in all stages of arterial aging and atherogenesis [10], [11], beginning early in life [10], [13], and in the critical CVD risk factor, hypertension [14].

Atherosclerosis and CVD are caused by a complex interaction of lifestyle factors (diet, energy balance, smoking, exercise) and inflammatory pathways. Elevated cholesterol and other risk factors predict less than half of heart attacks annually [15]. The acute phase inflammatory marker, C-reactive protein (CRP), often predicts future vascular events better than other risk factors [16], [17]. Indeed, individuals with lower than average cholesterol but high CRP have fewer heart attacks when given statins [18]. An estimated 25–30 million Americans fall into this category of low cholesterol but high CRP. Although CRP's role either as inflammatory marker or causal agent of vascular disease continues to be a subject of lively debate [19], [20], [21], it is increasingly used in clinical settings to help evaluate CVD risk.

Understanding the interaction of diet, energy balance, physical activity and inflammation is hampered by the populations studied: mainly well-fed in developed nations, or experimental animals fed ad libidum on diets that promote rapid growth. Both biomes contain few infectious pathogens and environmental inflammogens. In contrast, past human populations faced environments with strenuous physical demands, greater pathogen burden and low caloric diets. These environments characterize the lives of extant indigenous peoples with traditional lifestyles. Much of total mortality in those environments is by infections and parasites, rather than by the causes common in industrial populations, like chronic heart disease, diabetes and cancer [15], [22]. Thus, opposing risk conditions characterize such populations. On one hand, high infection rates over the life course should associate with greater lifetime inflammation exposure, and hence greater risk of atherosclerosis and CVD. On the other hand, high work effort, minimal obesity, and low fat intake should lower CVD risk.

Though arterial aging in the form of elastin fragmentation and medial fibrosis may be an inevitable outcome of aging, the role of atherosclerosis in adult morbidity before the 20^th century remains unclear. Studying arterial disease and CVD in indigenous populations is illuminating for three reasons. First, new light is shed on the roles and interactions of diet, exercise and inflammation on disease etiology. Second, subsistence populations with minimal medical access are characteristic of our evolutionary past, and may be informative about the role of vascular disease in the biology of aging during the long course of human evolution. Third, infectious disease is most prevalent in tropical regions of the developing world where the synergistic mix of risk factors and infectious causes are expected to bring a “gigantic epidemic of heart disease” in the coming decades [23].

Existing research provides contradictory findings and lacks important information on relevant variables. Traditional populations often show negligible CVD prior to acculturation to western diets and sedentary lifestyles [22], [24], [25]. Recently, however, Australian Aborigines and Pima Indians have acquired the highest prevalence of obesity and Type 2 diabetes in the world [26]. Higher rates of atherosclerosis, heart murmurs and other cardiac conditions among US soldiers in early 20^th century were linked to higher lifetime burden of infectious disease, particularly respiratory infections and rheumatic fever [27]. In one of the few non-western populations assessed for PAD, high prevalence was found among black South Africans [28], consistent with their extensive CVD mortality and morbidity. To date, no studies have measured the prevalence of PAD and other cardiovascular risk factors, such as hypertension, in an environmental context with chronically low energy status and high rates of infection.

Obesity is 8–10 times more common in the US than among Tsimane (Tables 1 and 2). The average BMI for Tsimane adults is 24, but 29 for US adults. Among Tsimane tobacco consumption is minimal, about 0.77±1.85 pack years for men and 0.14±0.78 for women. While 72% of Tsimane men and 14% of women reported occasional use of unfiltered cigarettes or tobacco from home gardens, most Tsimane even in their sixties have smoked less than 2 pack-years. By contrast, U.S. adults aged 60+ have smoked 24 (men) and 11 (women) years.

The main CVD risk factors greater among Tsimane than U.S. adults are markers of infection. Low HDL prevalence is also common, being about 3-fold greater among Tsimane, although the clinical significance of low HDL in energy-limited populations has not been established. Inflammatory markers (CRP, ESR, and WBC counts) were significantly higher among Tsimane (Tables 1 and 2).

Aside from sex, the strongest predictor of both SBP and DBP among Tsimane is BMI (standardized estimate = 0.178, 0.170, respectively). The magnitude of the effect, however, is not very large: an increase in BMI by 5 kg/m² increases SBP by 4 mm and DBP by 3 mm (Table 5). High total cholesterol and triglycerides are associated with high SBP and DBP, and LDL marginally predicts higher DBP. Similar patterns for these variables are found in the U.S., with smaller BMI and triglyceride effects. In the U.S., CRP significantly associates with higher SBP, and DBP when CRP values ≥10 mg/L are eliminated (β = 0.026, p<0.001).

Contrary to expectations, higher WBC and ESR associate with lower SBP and DBP among Tsimane, and CRP is not significant in the analyses. Restricting CRP<10 mg/L in the multiple regressions in Table 5, however, makes CRP a significant negative predictor of systolic pressure among Tsimane (β = −0.728, p = 0.026, n = 210), while in the U.S., CRP is a positive predictor of SBP (β = 0.579, p<0.001). Cigarette smoking among Tsimane and U.S. shows results contrary to standard prediction: higher tobacco consumption is associated with lower SBP and DBP (Table 5), but smoking loses significance among Tsimane in the multiple regression in Table 4.

Though our characterization of arterial disease is provisional pending ultrasonographic studies, our study provides evidence that chronic low-grade inflammation in the absence of several other risk factors is not a determinant of CVD in a subsistence population. Inflammation and infection may not accelerate arterial degeneration in the context of restricted caloric intake, parasitism, and daily physical activity that maintains low BMI. We observed low levels of atherosclerosis and associated CVD among Tsimane, suggesting that these conditions may have been rare throughout pre-industrial human history. However, as indigenous populations like the Tsimane rapidly acculturate to western lifestyles, rates of CVD among older adults may rise considerably. Transitioning populations exhibiting western lifestyles but relatively high pathogen load are likely to suffer the double burden of chronic and infectious disease morbidity and mortality [62], [63].

Informed consent was obtained for all protocols at three levels: 1) Gran Consejo Tsimane, the local Tsimane government organization that represents Tsimane interests and oversees all projects, 2) community officials and participants in village meetings, and 3) individual consent during medical visits and before each procedure. After explanation of a formal protocol by bilingual Tsimane assistants, consent forms were signed for literate participants, and verbal approval with fingerprint signature given for non-literate participants. Our consent procedures have been approved by the Institutional Review Boards at the Univ. of New Mexico, Univ. of California-Santa Barbara and Univ. of S. California.

Data were collected during annual medical exams of the Tsimane Health and Life History Project co-directed by MG and HK (www.unm.edu/~tsimane). Table 6 provides summary information on the sample population. Volunteers aged 40+ in 15 villages were sampled for ABI, blood pressure and anthropometry (2006 census population: 2,324 individuals; 350 were 40+, final sample n = 259, 133 or 52% male; 05–12/2007). Blood pressure was recorded from a larger sample of volunteers aged 20+ in 25 villages (eligible population 3,482; 1,298 are 20+; n = 1,262, 50% male; 11/2005–12/2007).

In-field blood analysis of nonfasting venous samples provided estimates of WBC and ESR. Blood measures exist for 234 ABI and 1,000 BP samples. CRP, total cholesterol, HDL and triglycerides were analyzed separately on a subset of serum samples by TriCore Laboratories (Albuquerque, NM). ABI exists for >110 of the 203 (age 40+), and blood pressure for 383 of the 427 (age 20+) samples with blood lipids, CRP and antibodies. Non-fasting LDL is estimated using the Friedewald formula (total cholesterol – HDL – triglycerides/5). CRP, ESR, and WBC are biomarkers of inflammation and infection.

Physicians measured ABI after training by HK and MG according to standard protocol by the American Heart Association. The patient lies supine with feet uncovered while brachial and ankle systolic pressure measurements are made using a SummitDoppler L150 ultrasound machine. Systolic pressure is first measured in the posterior and anterior tibial arteries and the higher of these is selected as the ankle measurement for each foot. The cuffs are inflated on the ankle to roughly 30 mm Hg above the systolic pressure, then followed by a slow release until the first audible sound of systolic pressure is heard. Systolic pressure is then measured in each arm and the highest is chosen as the brachial pressure. The ratio of the left and right ankle pressures to the brachial pressure is the left and right ABI. ABI <0.9 indicates PAD. ABI between 0.5 and 0.9 corresponds to intermittent claudication in the lower limbs, whereas values <0.5 are associated with more severe symptoms such as resting pain, severe occlusion and critical ischemia. ABI values >1.3 suggest calcification of arterial walls and noncompressible vessels, and are therefore also symptomatic of severe PAD.

Systolic (SBP) and diastolic (DBP) pressure were measured during each visit with a Welch Allyn Tycos Aneroid 5090 sphygmomanometer and Littman stethoscope. The systolic brachial pressure using the Doppler highly correlates with aneuroidal SBP (r = 0.896, p<0.0001). Cumulative experience smoking cigarettes was measured in cigarette pack years based on interviews. One pack year is equal to a pack of cigarettes smoked per day for one year. Bolivian physicians using bilingual Tsimane assistants diagnosed illnesses and trauma presented by patients on annual visits since 09/2002. Diagnoses from the International Classification of Disease (ICD-10) are grouped into gastrointestinal, respiratory, and other infections, over the two most recent exams 10/2005–12/2007.

Age estimates are derived from demographic interviews conducted with all individuals aged 18+ (n = 1,098) and from missionary records. Years of birth were assigned based on methodologies employed by researchers among the !Kung [64], Ache [65] and Hadza [66]. These include using known ages from written records, relative age lists, dated events, photo comparisons of people with known ages and cross-checking of information from independent interviews of kin. For example, Catholic missionaries have recorded the dates of 1,110 births among the Tsimane since 1952, many of the deaths occurring during the same period, and age estimates for an additional 120 individuals who were baptized as small children or as young adults during the early 1950's. In constructing relative age lists, multiple informants were used for each five year age grouping of individuals and inconsistencies were investigated and resolved. The photo comparison method used a sample of seventy photos of individuals with known ages. For older individuals, fifty photos of men and women from ages 50 through 75 were used. Each of these methods provides a roughly independent age estimate. When all estimates yield a date of birth within a 3-year range, the average was used unless one or two estimates were judged to be superior to the others. Further details are given in [1].

Tsimane health status was compared with NHANES data for the years 1999 to 2004 (N = 7,571 for adults age 40+, N = 14,213 for adults age 20+). The NHANES monitors the health and nutrition of a representative sample of the American noninstitutionalized population. Methods have been widely published and so are only briefly summarized here.

Lipid indicators include total, LDL and HDL cholesterol and triglycerides. Triglycerides and LDL were measured for approximately half of the sample that fasted for at least 6 hours. Total and HDL cholesterol and triglyceries were assayed using the Hitachi 704/717/912 Analyzer, Roche Diagnostics. Fasting LDL cholesterol was calculated using the Friedewald equation. CRP was determined by the latex-enhanced Behring Nephelometer. White blood cell differentials were determined using the Beckman Coulter® MAXM.

ABI was measured among the 40+ sample (N = 7,571) by trained technicians. People who had amputations, excessive obesity, or other conditions that inhibited examination were excluded. The procedure was the same as described for the Tsimane. Systolic and diastolic blood pressure was measured by physicians using a stethoscope and sphygmomanometer (Baumanometer® with a Calibrated® V-Lok® cuff, Latex Inflation Bulb, and an Air-Flo® Control Valve). SBP and DBP values are the average of three individual readings.

Multiple linear regression (PROC REG in SAS 9.1) was used for continuous values of ABI and blood pressure, as a function of demographic variables, risk factors and disease markers. Each risk factor is included separately in a baseline model that controls for sex, age and age² for ABI, and sex, age and sex*age for BP. These results are reported in Tables 3 and 5. Table 4 reports multiple regressions of ABI and BP that include BMI, CRP, cholesterol and cigarette smoking.

Post-hoc power analysis for multiple regression assuming a 0.05 alpha level was performed to assess whether the absence of significant effects in the Tsimane analyses was due to small sample size. Given the observed R², number of predictors and sample size, most regressions show power above 92%. Even considering the analysis of HDL on ABI (Table 3), where the sample size and R² were lowest, power was still 89.6%. Only in the regressions of diastolic BP (Table 5), where R²<0.08, did power reach as low as 70% (for HDL). The only analyses of Tsimane DBP that did not reveal statistically significant effects, however, also were not significant in the U.S. analyses, where power was 100%.