Vaughn A. Browne

Andean and Tibetan patterns of adaptation to high altitude.

High‐altitude hypoxia, or decreased oxygen levels caused by low barometric pressure, challenges the ability of humans to live and reproduce. Despite these challenges, human populations have lived on the Andean Altiplano and the Tibetan Plateau for millennia and exhibit unique circulatory, respiratory, and hematological adaptations to life at high altitude. We and others have identified natural selection candidate genes and gene regions for these adaptations using dense genome scan data. One gene previously known to be important in cellular oxygen sensing, egl nine homolog 1 (EGLN1), shows evidence of positive selection in both Tibetans and Andeans. Interestingly, the pattern of variation for this gene differs between the two populations. Continued research among Tibetan populations has identified statistical associations between hemoglobin concentration and single nucleotide polymorphism (SNP) genotype at EGLN1 and a second gene, endothelial PAS domain protein 1 (EPAS1).

High-end arteriolar resistance limits uterine artery blood flow and restricts fetal growth in preeclampsia and gestational hypertension at high altitude

The reduction in infant birth weight and increased frequency of preeclampsia (PE) in high-altitude residents have been attributed to greater placental hypoxia, smaller uterine artery (UA) diameter, and lower UA blood flow (Q(UA)). This cross-sectional case-control study determined UA, common iliac (CI), and external iliac (EI) arterial blood flow in Andeans residing at 3,600-4,100 m, who were either nonpregnant (NP, n = 23), or experiencing normotensive pregnancies (NORM; n = 155), preeclampsia (PE, n = 20), or gestational hypertension (GH, n = 12). Pregnancy enlarged UA diameter to ~0.62 cm in all groups, but indices of end-arteriolar vascular resistance were higher in PE or GH than in NORM. Q(UA) was lower in early-onset (≤34 wk) PE or GH than in NORM, but was normal in late-onset (>34 wk) illness. Left Q(UA) was consistently greater than right in NORM, but the pattern reversed in PE. Although Q(CI) and Q(EI) were higher in PE and GH than NORM, the fraction of Q(CI) distributed to the UA was reduced 2- to 3-fold. Women with early-onset PE delivered preterm, and 43% had stillborn small for gestational age (SGA) babies. Those with GH and late-onset PE delivered at term but had higher frequencies of SGA babies (GH=50%, PE=46% vs. NORM=15%, both P < 0.01). Birth weight was strongly associated with reduced Q(UA) (R(2) = 0.80, P < 0.01), as were disease severity and adverse fetal outcomes. We concluded that high end-arteriolar resistance, not smaller UA diameter, limited Q(UA) and restricted fetal growth in PE and GH. These are, to our knowledge, the first quantitative measurements of Q(UA) and pelvic blood flow in early- vs. late-onset PE in high-altitude residents.

Maternal PRKAA1 and EDNRA genotypes are associated with birth weight, and PRKAA1 with uterine artery diameter and metabolic homeostasis at high altitude

Low birth weight and intrauterine growth restriction (IUGR) increase the risk of mortality and morbidity during the perinatal period as well as in adulthood. Environmental and genetic factors contribute to IUGR, but the influence of maternal genetic variation on birth weight is largely unknown. We implemented a gene-by-environment study wherein we utilized the growth restrictive effects of high altitude. Multigenerational high-altitude residents (Andeans) are protected from altitude-associated IUGR compared with recent migrants (Europeans). Using a combined cohort of low- and high-altitude European and Andean women, we tested 63 single nucleotide polymorphisms (SNPs) from 16 natural selection-nominated candidate gene regions for associations with infant birth weight. We identified significant SNP associations with birth weight near coding regions for two genes involved in oxygen sensing and vascular control, PRKAA1 and EDNRA, respectively. Next, we identified a significant association for the PRKAA1 SNP with an intermediate phenotype, uterine artery diameter, which has been shown to be related to Andean protection from altitude-associated reductions in fetal growth. To explore potential functional relationships for the effect of maternal SNP genotype on birth weight, we evaluated the relationship between maternal PRKAA1 SNP genotype and gene expression patterns in general and, in particular, of key pathways involved in metabolic homeostasis that have been proposed to play a role in the pathophysiology of IUGR. Our observations suggest that maternal genetic variation within genes that regulate oxygen sensing, metabolic homeostasis, and vascular control influence fetal growth and birth weight outcomes and hence Andean adaptation to high altitude.

Uterine artery blood flow, fetal hypoxia and fetal growth

Evolutionary trade-offs required for bipedalism and brain expansion influence the pregnancy rise in uterine artery (UtA) blood flow and, in turn, reproductive success. We consider the importance of UtA blood flow by reviewing its determinants and presenting data from 191 normotensive (normal, n = 125) or hypertensive (preeclampsia (PE) or gestational hypertension (GH), n = 29) Andean residents of very high (4100–4300 m) or low altitude (400 m, n = 37). Prior studies show that UtA blood flow is reduced in pregnancies with intrauterine growth restriction (IUGR) but whether the IUGR is due to resultant fetal hypoxia is unclear. We found higher UtA blood flow and Doppler indices of fetal hypoxia in normotensive women at high versus low altitude but similar fetal growth. UtA blood flow was markedly lower in early-onset PE versus normal high-altitude women, and their fetuses more hypoxic as indicated by lower fetal heart rate, Doppler indices and greater IUGR. We concluded that, despite greater fetal hypoxia, fetal growth was well defended by higher UtA blood flows in normal Andeans at high altitude but when compounded by lower UtA blood flow in early-onset PE, exaggerated fetal hypoxia caused the fetus to respond by decreasing cardiac output and redistributing blood flow to help maintain brain development at the expense of growth elsewhere. We speculate that UtA blood flow is not only an important supply line but also a trigger for stimulating the metabolic and other processes regulating feto-placental metabolism and growth. Studies using the natural laboratory of high altitude are valuable for identifying the physiological and genetic mechanisms involved in human reproductive success.

Optic nerve sheath diameter and acute mountain sickness.

OBJECTIVE Increased intracranial pressure (ICP) may contribute to acute mountain sickness (AMS). Measuring optic nerve sheath diameter (ONSD) by ultrasound (US) is a noninvasive technique to detect elevated ICP, and increased ONSD has been associated with AMS. We hypothesized that ONSD would increase with acute, rapid ascent to 4300 m and that increased ONSD would be associated with symptoms of AMS. We further hypothesized that treatment with oxygen at 4300 m would reduce symptoms and ONSD. METHODS A cohort study was performed comparing US measurement of ONSD in healthy subjects at 1400 m and 18 hours after rapid ascent to 4300 m, both before and after oxygen treatment and between subjects with and without AMS (Lake Louise Score ≥3). RESULTS Among 57 subjects, 29 (51%) experienced AMS after rapid ascent to 4300 m. In subjects without AMS, mean ONSD did not increase at 4300 m. In subjects with AMS, mean ONSD increased at 4300 m and was higher than in those without AMS. Treatment with oxygen lowered mean ONSD in subjects with AMS but not in those without AMS. Individual responses to altitude and oxygen varied greatly within groups, and the relationship between ONSD and AMS symptoms was weak. CONCLUSIONS In this controlled study, mean ONSD increased in subjects with AMS at high altitude. However, individual variation was high, and most ONSD values were below the clinical threshold for raised ICP. Observed differences were small, of questionable clinical importance, and within the range of precision of the US machine. Overall, our data do not support a role for increased ICP in mild to moderate AMS.

Potential role for elevated maternal enzymatic antioxidant status in Andean protection against altitude-associated SGA.

Oxidative stress has been implicated in the uteroplacental ischemia characteristic of preeclampsia and small-for-gestational-age (SGA) birth, both of which are more common at high (>2500 m) vs low altitude. Since Andeans are protected relative to Europeans from the altitude-associated rise in SGA, we asked whether alterations in maternal antioxidant status or oxidative stress contributed to their protection. Enzymatic antioxidant (erythrocyte catalase and superoxide dismutase [SOD]) activity and a plasma marker of lipid peroxidation (8-iso-PGF2α) were measured during pregnancy and in the non-pregnant state in Andean or European residents of low (400 m) or high altitude (3600–4100 m). Pregnancy and altitude increased catalase and/or SOD activity to a greater extent in Andeans than Europeans. 8-iso-PGF2α levels were independent of altitude and pregnancy. SOD was lower in mothers of SGA infants at weeks 20 and 36. Our findings are consistent with the possibility that elevated enzymatic antioxidant activity contributes to Andean protection against altitude-associated SGA.

Do cytokines contribute to the Andean-associated protection from reduced fetal growth at high altitude?

Pro- versus anti-inflammatory cytokine balance is important for successful pregnancy. Chronic hypoxia alters cytokine levels and increases the frequency of fetal growth restriction (FGR). Multigenerational Andean (AND) versus shorter duration European (EUR) high-altitude (HA) residents are protected from altitude-associated FGR. To address whether ancestry group differences in cytokine levels were involved, we conducted serial studies in 56 low-altitude ([LA]; 400 m; n = 29 AND and n = 27 EUR) and 42 HA residents (3600-4100 m; n = 19 ANDs and n = 23 EURs). Pregnancy raised pro- (interleukin 1β [IL-1β]) and anti- (IL-10) inflammatory cytokines and HA lowered IL-6 and tumor necrosis factor-α (TNF-α) near term. There were no ancestry group differences in cytokine levels at any time, but HA reduced IL-1β in ANDs only near term. Higher IL-1β levels correlated with uterine artery (UA) blood flow at 20 weeks in ANDs at HA, suggesting that IL-1β may play a role in AND protection from altitude-associated reductions in fetal growth.

Do Anti-angiogenic or Angiogenic Factors Contribute to the Protection of Birth Weight at High Altitude Afforded by Andean Ancestry?

Objective: This prospective study was designed to determine whether variation in angiogenic (placental growth factor [PlGF]) and/or anti-angiogenic (soluble fms-like tyrosine kinase [sFlt-1]) factors contribute to the protective effect of highland ancestry (Andean) from altitude-associated reductions in fetal growth. Study design: Plasma sFlt-1 and PlGF levels, uterine artery (UA) blood flow, and fetal biometry were determined in low-altitude (400 m; Andean n = 27, European n = 28) and high-altitude (3600 m; Andean n = 51, European n = 44) residents during pregnancy (20 and 36 weeks) and 4 months postpartum. Results: High-altitude decreased sFlt-1 levels in both groups, Andeans had lower sFlt-1, comparable PlGF, lower sFlt-1/PlGF ratios, and higher UA blood flow throughout pregnancy relative to Europeans. Altitude decreased birth weight in Europeans but not Andeans. In high-altitude Europeans sFlt-1/PlGF and sFlt-1 levels were negatively associated with UA diameter and birth weight, respectively. Conclusions: Lower sFlt-1 and sFlt-1/PLGF ratio may contribute to or result from variations in maternal vascular adaptation to pregnancy between Andean and Europeans at high altitude. Subsequently, these effects could potentially influence ancestry-associated differences in birth weight.

Effects of long-term high-altitude hypoxia and troponin I phosphorylation on cardiac myofilament calcium responses in Fetal and nonpregnant sheep

Objective: We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep. Methods: Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state. Results: In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significanlty less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left venitricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small. Conclusion: In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphophatase activity observed previously. The larger decrease in tension after PKA phosphroylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.

Inhibition of peroxisome proliferator-activated receptor γ: a potential link between chronic maternal hypoxia and impaired fetal growth

Chronic exposure to hypoxia raises the risk of pregnancy disorders characterized by maternal vascular dysfunction and diminished fetal growth. In an effort to identify novel pathways for these hypoxia‐related effects, we assessed gene expression profiles of peripheral blood mononuclear cells (PBMCs) obtained from 43 female, high‐altitude or sea‐level residents in the nonpregnant state or during pregnancy (20 or 36 wk). Hypoxia‐related fetal growth restriction becomes apparent between 25 and 29 wk of gestation and continues until delivery. Our sampling strategy was designed to capture changes occurring before (20 wk) and during (36 wk) the time frame of slowed fetal growth. PBMC gene expression profiles were generated using human gene expression microarrays and compared between altitudes. Biological pathways were identified using pathway analysis. Modest transcriptional differences were observed between altitudes in the nonpregnant state. Of the genes that were differentially expressed at high altitude vs. sea level during pregnancy (20 wk: 59 probes mapped to 41 genes; 36 wk: 985 probes mapped to 700 genes), several are of pathological relevance for fetal growth restriction. In particular, transcriptional changes were consistent with the negative regulation of peroxisome proliferator‐activated receptor γ (PPARγ) at high altitude; such effects were accompanied by reduced birth weight (P <0.05) and head circumference (P <0.01) at high altitude vs. sea level. Our findings indicate that chronic exposure to hypoxia during pregnancy alters maternal gene expression patterns in general and, in particular, expression of key genes involved in metabolic homeostasis that have been proposed to play a role in the pathophysiology of fetal growth restriction.—Julian, C. G., Yang, I. V., Browne, V. A., Vargas, E., Rodriguez, C., Pedersen, B. S., Moore, L. G., Schwartz, D. A. Inhibition of peroxisome proliferator‐activated receptor γ: a potential link between chronic maternal hypoxia and impaired fetal growth. FASEB J. 28, 1268–1279 (2014). www.fasebj.org