Ulf J. Eriksson

Increased Incidence of Congenital Malformations in the Offspring of Diabetic Rats and Their Prevention by Maternal Insulin Therapy

Intensive care of the pregnant mother with diabetes has dramatically decreased the incidence of diabetic fetopathy. The persistently high rate of fetal and neonatal mortality in diabetic pregnancies is nowadays mainly due to the increased incidence of serious congenital malformations. However, attempts to elucidate the precise teratogenic mechanisms have been sparse, presumably because of a lack of relevant animal models. In the present study we recorded the incidence and types of skeletal malformations in live offspring of normal rats and in rats made diabetic with the B-cytotoxic agent streptozotocin (SZ) at least 2 wk before conception. In some of the diabetic animals insulin treatment was begun 1 wk after the SZ injection and continued throughout pregnancy. In addition, the fetal development was followed by assessing the calcification of the skeleton on gestational days 20 and 22 with the aid of Alazarin Red S staining. Manifest diabetes in the pregnant rat induced a decrease in fetal weight and viability and marked retardation of skeletal maturation. In addition, about 20% of 135 viable fetuses showed skeletal malformations comprising either micrognathia or caudal dysgenesis. These defects were not found in 314 offspring of the control rats. Only two cases of caudal dysgenesis and none of micrognathia were detected among 233 offspring of the insulin-treated rats. The present data underscore the importance of a strict differentiation in the offspring of the diabetic rat between transient development retardations and true malformations. They also demonstrate that correction of the maternal glucose intolerance is crucial for preventing the fetus from developing skeletal malformations. Altogether the data suggest that fetal malformations in the diabetic rat are attributable either to the hyperglycemia as such or to some accompanying metabolic consequence of insulin deficiency.

Protection by free oxygen radical scavenging enzymes against glucose-induced embryonic malformations in vitro.

SummaryThis study addresses the possibility that the teratogenic effects of a diabetic pregnancy are associated with increased embryonic activities of free oxygen radicals. Rat embryos were cultured in 50 mmol/l glucose for 48 h and subsequently showed pronounced growth retardation and severe malformations. The enzyme inducer citiolone and the free oxygen radical scavenging enzymes Superoxide dismutase, catalase and glutathione peroxidase protected against the disturbed growth and development of the embryos at 50 mmol/l glucose when added to the culture media. Enzymatic measurements indicated that citiolone induced an increased activity of superoxide dismutase in the embryonic tissues and that the added enzymes were taken up by both the yolk sac and the embryo proper. The protection against embryonic maldevelopment was thus conferred by agents that increased the free oxygen radical scavenging capacity of the embryonic tissues. The results suggest that a high glucose concentration in vitro causes embryonic dysmorphogenesis by generation of free oxygen radicals. An enhanced production of such radicals in embryonic tissues may be directly related to the increased risk of congenital malformations in diabetic pregnancy.

Diabetes and Embryonic Malformations: Role of Substrate-Induced Free-Oxygen Radical Production for Dysmorphogenesis in Cultured Rat Embryos

The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy. Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, 3 mM PYR, 10 mM HBT, or 3 mM KIC. Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations. The deleterious effects on embryonic development by all agents were alleviated by addition of SOD to the culture media, which yielded increased enzyme activity in the embryos and their membranes. The endogenous SOD activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium. Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC. These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment. In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload. This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women. Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.

Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease

Excess lipid accumulation in peripheral tissues is a key feature of many metabolic diseases. Therefore, techniques for imaging and quantifying lipids in various tissues are important for understanding and evaluating the overall metabolic status of a research subject. Here we present a protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues. The method allows for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment. Furthermore, the procedure described here is well suited for the comparison of different metabolically challenged animal models. As an example, we include data on muscular and hepatic lipid accumulation in diet-induced and genetically induced diabetic mice. The experimental description presents details for optimal staining of lipids using ORO, including tissue collection, sectioning, staining, imaging and measurements of tissue lipids, in a time frame of less than 2 d.

Vascular Endothelial Growth Factor-B–Deficient Mice Display an Atrial Conduction Defect

Background—Vascular endothelial growth factors (VEGFs) and their receptors are essential regulators of vasculogenesis and angiogenesis in both embryos and adults. One of the factors with a still unknown physiological function is VEGF-B, which is expressed in many tissues, including the heart. Methods and Results—Mice carrying a targeted deletion in the VEGF-B gene were developed. In VEGF-B−/− animals, no gross abnormalities were observed in organs that normally show high expression of VEGF-B, such as the heart, muscle, and kidney. Analysis of heart function by ECG showed that adult VEGF-B−/− mice have an atrial conduction abnormality characterized by a prolonged PQ interval. VEGF- or basic fibroblast growth factor–induced corneal angiogenesis was similar in normal and VEGF-B−/− mice. Conclusions—VEGF-B seems to be required for normal heart function in adult animals but is not required for proper development of the cardiovascular system either during development or for angiogenesis in adults.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate

Platelet-derived growth factor (PDGF) receptor signaling participates in different processes in solid tumors, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. In the present study, the B16 mouse melanoma tumor model was used to investigate the functional consequences of paracrine PDGF stimulation of host-derived cells. Production of PDGF-BB or PDGF-DD by tumor cells was associated with an increased tumor growth rate. Characterization of tumors revealed an increase in pericyte abundance in tumors derived from B16 cells producing PDGF-BB or PDGF-DD. The increased tumor growth rate associated with PDGF-DD production was not seen in mice expressing an attenuated PDGF beta-receptor and was thus dependent on host PDGF beta-receptor signaling. The increased pericyte abundance was not associated with an increased tumor vessel density. However, tumor cell apoptosis, but not proliferation, was reduced in tumors displaying PDGF-induced increased pericyte coverage. Our findings thus demonstrate that paracrine PDGF production stimulates pericyte recruitment to tumor vessels and suggest that pericyte abundance influences tumor cell apoptosis and tumor growth.

Paracrine Signaling by Platelet-Derived Growth Factor-CC Promotes Tumor Growth by Recruitment of Cancer-Associated Fibroblasts

Cancer results from the concerted performance of malignant cells and stromal cells. Cell types populating the microenvironment are enlisted by the tumor to secrete a host of growth-promoting cues, thus upholding tumor initiation and progression. Platelet-derived growth factors (PDGF) support the formation of a prominent tumor stromal compartment by as of yet unidentified molecular effectors. Whereas PDGF-CC induces fibroblast reactivity and fibrosis in a range of tissues, little is known about the function of PDGF-CC in shaping the tumor-stroma interplay. Herein, we present evidence for a paracrine signaling network involving PDGF-CC and PDGF receptor-alpha in malignant melanoma. Expression of PDGFC in a mouse model accelerated tumor growth through recruitment and activation of different subsets of cancer-associated fibroblasts. In seeking the molecular identity of the supporting factors provided by cancer-associated fibroblasts, we made use of antibody arrays and an in vivo coinjection model to identify osteopontin as the effector of the augmented tumor growth induced by PDGF-CC. In conclusion, we establish paracrine signaling by PDGF-CC as a potential drug target to reduce stromal support in malignant melanoma.

Placental Growth Factor and Soluble FMS- Like Tyrosine Kinase-1 in Early-Onset and Late-Onset Preeclampsia

OBJECTIVE: To estimate whether alterations in plasma levels of the proangiogenic proteins placental growth factor (PlGF) and vascular endothelial growth factor-A (VEGF-A), and the antiangiogenic protein soluble fms-like tyrosine kinase-1 (sFlt1) were more pronounced in early-onset than in late-onset preeclampsia. METHODS: A cross-sectional study was conducted to estimate the levels of sFlt1, PlGF, and VEGF-A in plasma in a control group of nonpregnant women, in an early control group of women at 24–32 weeks of gestation, in a late control group of women at 36–42 weeks of gestation, and in cases of women with early-onset (before 32 weeks of gestation) and late-onset (after 35 weeks of gestation) preeclampsia. RESULTS: Women with early-onset preeclampsia had a 43 times higher median plasma sFlt1 level than early controls (P<.001). Women with late-onset preeclampsia had a three times higher median plasma sFlt1 level than late controls (P<.001). Women with early-onset preeclampsia had a 21 times lower median plasma PlGF level than early controls (P<.001). Women with late-onset preeclampsia had a five times lower median plasma PlGF level than late controls (P=.01). The median level of VEGF-A in plasma was less than 15 pg/mL in all groups of pregnant women. CONCLUSION: Both early- and late-onset preeclampsia are associated with altered plasma levels of sFlt1 and PlGF. The alterations are more pronounced in early-onset rather than in late-onset disease. LEVEL OF EVIDENCE: II

Prevention of diabetes-associated embryopathy by overexpression of the free radical scavenger copper zinc superoxide dismutase in transgenic mouse embryos

OBJECTIVES It has recently been suggested that oxygen free radicals are involved in the high incidence of fetal dysmorphogenesis that is associated with diabetic pregnancies. The purpose of the current investigation was to study the effect of copper zinc superoxide dismutase, a free radical scavenging enzyme, on the prevention of diabetes-associated embryopathy in mice. STUDY DESIGN Mice used in this study were either transgenic, bearing the human copper zinc superoxide dismutase gene, or nontransgenic controls. Diabetes was generated by streptozotocin administration on days 6 and 7 of gestation. Hyperglycemia developed on day 8 and was maintained through day 10 (critical period of organogenesis). On day 10 fetuses were examined for external anomalies, and their crown-rump lengths and deoxyribonucleic acid content were determined. RESULTS Induction of maternal diabetes produced a significant reduction in mean crown-rump length of control embryos (4.48 +/- 0.7 mm vs 3.65 +/- 0.6 mm, p = 0.0001), whereas transgenic embryos were not affected (4.72 +/- 0.6 mm vs 4.45 +/- 0.8 mm, p > 0.05). After induction of diabetes fetal loss and malformation rates were significantly higher in control embryos (6.0% vs 23.8% and 8.4% vs 16.5%, respectively). Transgenic embryos were practically unaffected by diabetes and showed fetal loss and malformation rates of 5.9% and 4.4%, respectively, after induction of diabetes. CONCLUSIONS Elevated levels of copper zinc superoxide dismutase, a key enzyme in the metabolism of free oxygen radicals, elicit a protective effect against diabetes-associated embryopathy.