Role of Macrophages in Developmental Programming of Pancreatic β‐Cell Area in Offspring of Gestational Hypertension

Abstract

Preeclampsia is a pregnancy specific complication characterized by new‐onset maternal hypertension and a high incidence of intrauterine growth restriction (IUGR), both associated with negative outcomes for offspring. Our previous studies indicated that IUGR in conjunction with maternal hypertension in the rat Reduced Uteroplacental Perfusion Pressure (RUPP) model of chronic placental ischemia results in female offspring with reduced pancreatic β‐cell area compared to sham pregnancies at embryonic day 19, and this decrease in β‐cell area persists to postnatal day 13 (PD13). Pancreatic β‐cell area, set very early in life, can influence whether an individual develops type 2 diabetes (T2D). Consistent with reduced β‐cell area, previous studies of others have demonstrated oral glucose intolerance in 6‐12 month RUPP offspring. T2D is associated with increased macrophages in the pancreas, and we previously reported increased message for macrophage marker CD68 is associated with reduced β‐cell area in islets of female PD13 RUPP animals. Thus, we hypothesized that macrophages are crucial to the reduction in β‐cell area in RUPP offspring. To test this hypothesis, we determined if postnatal depletion of macrophages rescued the β‐cell area. Chronic placental ischemia was induced in timed‐pregnant Sprague‐Dawley rats using the RUPP model by surgical placement of clips on abdominal aorta and uterine arteries on gestation day 14 of a 21‐day gestation. Female offspring were intraperitoneally injected with control liposomes or clodronate liposomes (56 and 28 mg clodronate/kg on PD2 and PD9, respectively) to deplete macrophages. On PD13, offspring were euthanized and pancreata fixed in formalin for paraffin embedding and determination of β‐cell area. Other pancreata were perfused with collagenase and digested for islet isolation by hand‐picking. β‐cell/pancreas area was determined by immunofluorescence and image analysis. Female RUPP animals treated with control liposomes had significantly reduced β‐cell area compared to sham offspring treated with control liposomes (0.0156 vs 0.0242 β‐cell/pancreas area, n=5, p<0.05). Notably, β‐cell area of sham or RUPP offspring treated with clodronate liposomes was not different (0.0221 vs 0.0225 β‐cell/pancreas area, n= 6‐7) from sham animals treated with control liposomes (0.0242 β‐cell/pancreas area, n=5). Thus, clodronate liposome treatment rescued the defect in β‐cell area of female RUPP offspring compared to RUPP animals treated with control liposomes. Message for CD68 in islets was significantly decreased following clodronate treatment, suggesting depletion in the islet. In addition, macrophages were significantly depleted in peritoneal lavage, spleen and islets as determined by flow cytometry. Total macrophages in lavage decreased from 7.1 x 10^5 to 0.05 x 10^5 cells following clodronate treatment. Thus, our studies suggest macrophages are critical for the reduction in β‐cell area in RUPP offspring following chronic placental ischemia. Future studies will explore the influence of apoptosis, proliferation, β‐cell size and number on reduction in β‐cell area.