Effects of dietary soy protein on iliac and carotid artery atherosclerosis and gene expression in male monkeys

Abstract

Male cynomolgus macaques (n=91) consumed an isoflavone (IF)-free, atherogenic control diet containing casein/lactalbumin for five months, then were randomized to three groups: Control (n=30) continued on the control diet; Low IF (n=30) received a mixture of unmodified and IF-depleted soy protein isolate (SPI) (0.94 mg IF/g protein, approximating a human intake of 75 mg/day); and High IF (n=31) received unmodified SPI (1.88 mg IF/g protein, approximating a human intake of 150mg/ day) for 31 months. Iliac and carotid artery atherosclerosis, and arterial and hepatic mRNA transcripts related to inflammation and estrogen receptors (ER) were measured. Trend analysis identified a significant inverse relationship between dietary IF content and plaque area in the iliac (p<0.05) but not carotid arteries (p>0.13). No significant effect of diet on inflammatory gene or estrogen receptor expression was observed. Plaque area was positively correlated with the mRNA transcript levels for arterial MCP-1, ICAM-1, and the macrophage marker CD68 (all r>0.25, p<0.03), and negatively correlated with Erα and ERβ (all r<−0.23, p<0.03). Coronary artery plaque area appeared to be more closely associated with gene expression patterns of the iliac arteries than the carotid arteries. The data suggests benefits of dietary soy on atherosclerotic plaque development in males may be mediated through inflammation-independent pathways. The negative associations of arterial ERα expression with atherosclerosis lend support to a mechanistic role for estrogen receptors in atherosclerosis susceptibility which merits further study.

Differential effects of estradiol on carotid artery inflammation when administered early versus late after surgical menopause

“…The carotid arteries were removed, cleaned of adherent connective tissues, opened longitudinally, and sectioned. The common carotid arteries were subdivided into individual segments and processed as previously described for iliac arteries 22 . Briefly, adjacent segments were flash frozen in liquid nitrogen for RNA isolation and fixed in 4% paraformaldehyde for histologic examination.…”

“…Briefly, paraffin blocks were sectioned at 5 μm and stained with Verhoeff Van Gieson as well as hematoxylin and eosin for assessment of tissue histology. Plaque area (mm2) of the carotid artery sections were determined by computer assisted histomorphometric methods using Image Pro Plus software 22 (Media Cybernetics, Inc. Silver Springs, MD).…”

“…Total RNA (2 μg) was reverse transcribed to generate cDNA archive using a high-capacity cDNA archive kit (Applied Biosystems). Quantitative real-time reverse transcriptase-polymerase chain reaction (PCR) assays were performed on an ABI Prism 7500 FAST system using macaque-specific TaqMan FAM-MGB primer-probe assays for CD3, CD4, CD8, CD25, CD56 or neural cell adhesion molecule-1 (NCAM-1), CD83, CD163, forkhead box P3 (FoxP3), interleukin-2 (IL-2), interleukin-4 (IL-4), IL-10, tumor necrotic factor-alpha (TNF-α), interferon-γ (IFN-γ), matrix metalloproteinase-9 (MMP-9), MCP-1, chemokine (C-C motif) receptor 6 (CCR6), ER-α and ER-β or human-specific CD68, collagen type I (Col1), and smooth muscle actin (ACTA2) (Applied Biosystems) 8, 22 . Individual PCR reactions were performed using cDNA generated from 27 ng total RNA.…”

Molecular and cellular profiling of acute responses to total body radiation exposure in ovariectomized female cynomolgus macaques

“…Quantitative real-time polymerase chain reaction (RTPCR) was used to evaluate the inflammatory cell content of thymus, spleen, and lymph node using previously described methods (Sophonsritsuk et al 2013; Walker et al 2008a, 2008b). Levels of mRNA transcripts for a specific marker associated with macrophages cluster of differentiation 68 (CD68), and four markers of T cells T-cell receptor beta, cluster of differentiation 3, 4 and 8 (TCRB, CD3, CD4, CD8) were measured in order to define the cellular composition of the tissue.…”

“…RNA quality was assessed by intensity of 28S and 18S ribosomal RNA bands on an Agilent Microanalyzer. Quantitative Taqman ® RT-PCR assays employing cynomolgus-specific primers and Fam-labeled Taqman ® MGB probes were used to quantitate mRNA steady-state levels (Sophonsritsuk et al, 2013; Walker et al, 2008a, 2008b). Aliquots of purified total RNA (~ 1–10 ug RNA) were reverse transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) in combination with random hexamers to generate cDNA from which real time RT-PCR quantitative analyses were performed.…”