Fetal sex modulates placental microRNA expression, potential microRNA-mRNA interactions, and levels of amino acid transporter expression and substrates: INFAT study subpopulation analysis of n-3 LCPUFA intervention during pregnancy and associations with offspring body composition.

BACKGROUND: Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition. RESULTS: We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks' gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age. CONCLUSIONS: Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.

Dietary n-3 long-chain polyunsaturated fatty acids upregulate energy dissipating metabolic pathways conveying anti-obesogenic effects in mice.

BACKGROUND: We previously reported on the anti-obesogenic and anti-inflammatory effects associated with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in our diet-induced obesity (DIO) mouse model. Two isocaloric high-fat diets (HFDs; 48 kJ% fat), HFD (HF) and n-3 LCPUFA-enriched HFD (HF/n-3), and a control diet (C; 13 kJ% fat) were used. The underlying mechanisms however have largely remained unclear. Here, we assessed whether the reduced fat mass reflected n-3 LCPUFA-induced expression changes in lipid metabolism of the intestine, liver, and interscapular brown adipose tissue (iBAT), as well as increased iBAT thermogenic capacity. METHODS: For HF/n-3, saturated and monounsaturated fatty acids were partially substituted by n-3 LCPUFA eicosapentaenoic acid and docosahexaenoic acid to achieve a balanced n-6/n-3 PUFA ratio (0.84) compared to the unbalanced ratios of HF (13.5) and C (9.85). Intestine, liver and iBAT from male C57BL/6 J mice, fed defined soybean/palm oil-based diets for 12 weeks, were further analysed. Gene and protein expression analyses, immunohistochemistry and correlation analyses for metabolic interactions were performed. RESULTS: Compared to HF and C, our analyses suggest significantly diminished de novo lipogenesis (DNL) and/or increased hepatic and intestinal fatty acid oxidation (ω-oxidation and peroxisomal ß-oxidation) in HF/n-3 mice. For iBAT, the thermogenic potential was enhanced upon HF/n-3 consistent with upregulated expression for uncoupling protein-1 and genes involved in mitochondrial biogenesis. In addition, a higher capacity for the supply and oxidation of fatty acids was observed and expression and correlation analyses indicated a coordinated regulation of energy metabolism and futile cycling of triacylglycerol (TAG). Moreover, HF/n-3 significantly increased the number of anti-inflammatory macrophages and eosinophils and significantly enhanced the levels of activated AMP-activated protein kinase α (AMPKα), peroxisome proliferator-activated receptor α (PPARα) and fibroblast growth factor 21 (FGF21). CONCLUSIONS: Our data suggest that by targeting transcriptional regulatory pathways, AMPKα, and FGF21 as potential mediators, HF/n-3 activated less efficient pathways for energy production, such as peroxisomal ß-oxidation, increased ATP consumption upon the induction of futile cycling of TAG, and additionally increased the thermogenic and oxidative potential of iBAT. Therefore, we consider n-3 LCPUFA as the potent inducer for upregulating energy dissipating metabolic pathways conveying anti-obesogenic effects in mice.

Human placental transcriptome shows sexually dimorphic gene expression and responsiveness to maternal dietary n-3 long-chain polyunsaturated fatty acid intervention during pregnancy.

BACKGROUND: Previously we have examined the effect of maternal dietary n-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation during pregnancy on offspring fat mass. Considering the involvement of the placenta in fetal programming, we aimed to analyze the sex-specific gene expression in human term placenta and its response to the n-3 LCPUFA intervention, as well as their correlations to offspring adiposity. RESULTS: Placental gene expression was assessed in a control and n-3 LCPUFA intervention group by DNA microarrays, biological pathway analyses and RT-qPCR validation. Expression data were correlated with sex steroid hormone levels in placenta and cord plasma, and offspring anthropometric data. Transcriptome data revealed sexually dimorphic gene expression in control placentas per se, whereas in intervention placentas sex-specific expression changed, and more n-3 LCPUFA-regulated genes were found in female than male placentas. Sexually dimorphic gene expression and n-3 LCPUFA-responsive genes were enriched in the pathway for cell cycle and its associated modulator pathways. Significant mRNA expression changes for CDK6, PCNA, and TGFB1 were confirmed by RT-qPCR. CDK6 and PCNA mRNA levels correlated with offspring birth weight and birth weight percentiles. Significantly reduced placental estradiol-17ß/testosterone ratio upon intervention found in female offspring correlated with mRNA levels for the 'Wnt signaling' genes DVL1 and LRP6. CONCLUSIONS: Overall, human placentas show sexually dimorphic gene expression and responsiveness to maternal n-3 LCPUFA intervention during pregnancy with more pronounced effects in female placentas. The absence of correlations of analyzed placental gene expression with offspring adipose tissue growth in the first year is not mutually exclusive with programming effects, which may manifest later in life, or in other physiological processes.

Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced ß-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid ß-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Effect of maternal obesity with and without gestational diabetes on offspring subcutaneous and preperitoneal adipose tissue development from birth up to year-1.

BACKGROUND: Maternal obesity and gestational diabetes mellitus (GDM) may independently influence offspring fat mass and metabolic disease susceptibility. In this pilot study, body composition and fat distribution in offspring from obese women with and without GDM and lean women were assessed within the 1st year of life, and maternal and newborn plasma factors were related to offspring adipose tissue distribution. METHODS: Serum and plasma samples from pregnant obese women with (n = 16) or without (n = 13) GDM and normoglycemic lean women (n = 15) at 3rd trimester and offspring cord plasma were used for analyzing lipid profiles, insulin and adipokine levels. At week-1 and 6, month-4 and year-1, offspring anthropometrics and skinfold thickness (SFT) were measured and abdominal subcutaneous (SCA) and preperitoneal adipose tissue (PPA) were determined by ultrasonography. RESULTS: Cord insulin was significantly increased in the GDM group, whereas levels of cord leptin, total and high molecular weight (HMW) adiponectin were similar between the groups. Neonates of the GDM group showed significantly higher SFT and fat mass until week-6 and significantly increased SCA at week-1 compared to the lean group that persisted as strong trend at week-6. Interestingly, PPA in neonates of the GDM group was significantly elevated at week-1 compared to both the lean and obese group. At month-4 and year-1, significant differences in adipose tissue growth between the groups were not observed. Multiple linear regression analyses revealed that cord insulin levels are independently related to neonatal PPA that showed significant relation to PPA development at year-1. Maternal fasted C-peptide and HMW adiponectin levels at 3rd trimester emerged to be determinants for PPA at week-1. CONCLUSION: Maternal pregravid obesity combined with GDM leads to newborn hyperinsulinemia and increased offspring fat mass until week-6, whereas pregravid obesity without GDM does not. This strongly suggests the pivotal role of GDM in the adverse offspring outcome. Maternal C-peptide and HMW adiponectin levels in pregnancy emerge to be predictive for elevated PPA in newborns and might be indicative for the obesity risk at later life. Altogether, the findings from our pilot study warrant evaluation in long-term studies. TRIAL REGISTRATION: German Clinical Trials Register DRKS00004370.

Effect of reducing the n-6/n-3 fatty acid ratio on the maternal and fetal leptin axis in relation to infant body composition.

OBJECTIVE: To investigate the effect of reducing the n-6/n-3 fatty acid ratio in maternal nutrition on the maternal and cord blood leptin axis and their association with infant body composition up to 2 years. DESIGN AND METHODS: 208 healthy pregnant women were randomized to either a dietary intervention to reduce the n-6/n-3 fatty acid ratio from 15th week of gestation until 4 months postpartum or a control group. Leptin, soluble leptin receptor and free leptin index were determined in maternal and cord plasma and related to infant body composition assessed by skinfold thicknesses up to 2 years. RESULTS: The intervention had no effect on either the maternal or fetal leptin axis. Maternal leptin in late pregnancy was inversely related to infant weight and lean body mass (LBM) up to 2 years, after multiple adjustments. Cord leptin was positively related to weight, body fat, and LBM at birth, and inversely associated with weight, BMI, fat mass, and LBM at 2 years and weight gain up to 2 years. The contribution of cord leptin to infant outcomes was overall stronger compared with maternal leptin. CONCLUSIONS: Both, maternal and fetal leptin were associated with subsequent infant anthropometry with a greater impact of fetal leptin.

Breast milk fatty acid profile in relation to infant growth and body composition: results from the INFAT study.

BACKGROUND: There is some evidence that the n-6/n-3 long-chain polyunsaturated fatty acids (LCPUFAs) ratio in early nutrition, and thus in breast milk, could influence infant body composition. METHODS: In an open-label randomized controlled trial (RCT), 208 healthy pregnant women were allocated to a dietary intervention (supplementation with 1,200 mg n-3 LCPUFAs per day and instructions to reduce arachidonic acid (AA) intake) from the 15th wk of gestation until 4 mo of lactation or to follow their habitual diet. Breast milk LCPUFAs at 6 wk and 4 mo postpartum were related to infant body composition assessed by skinfold thickness (SFT) measurements and ultrasonography during the first year of life. RESULTS: Dietary intervention significantly reduced breast milk n-6/n-3 LCPUFAs ratio. In the whole sample, early breast milk docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 LCPUFAs at 6 wk postpartum were positively related to the sum of four SFT measurements at age 1. Breast milk AA and n-6 LCPUFAs at 6 wk postpartum were negatively associated with weight, BMI, and lean body mass (LBM) up to 4 mo postpartum. CONCLUSION: Breast milk n-3 LCPUFAs appear to stimulate fat mass growth over the first year of life, whereas AA seems to be involved in the regulation of overall growth, especially in the early postpartum period.

Integrins modulate the infection efficiency of West Nile virus into cells.

The underlying mechanisms allowing West Nile virus (WNV) to replicate in a large variety of different arthropod, bird and mammal species are largely unknown but are believed to rely on highly conserved proteins relevant for viral entry and replication. Consistent with this, the integrin αvß3 has been proposed lately to function as the cellular receptor for WNV. More recently published data, however, are not in line with this concept. Integrins are highly conserved among diverse taxa and are expressed by almost every cell type at high numbers. Our study was designed to clarify the involvement of integrins in WNV infection of cells. A cell culture model, based on wild-type and specific integrin knockout cell lines lacking the integrin subunits αv, ß1 or ß3, was used to investigate the susceptibility to WNV, and to evaluate binding and replication efficiencies of four distinct strains (New York 1999, Uganda 1937, Sarafend and Dakar). Though all cell lines were permissive, clear differences in replication efficiencies were observed. Rescue of the ß3-integrin subunit resulted in enhanced WNV yields of up to 90 %, regardless of the virus strain used. Similar results were obtained for ß1-expressing and non-expressing cells. Binding, however, was not affected by the expression of the integrins in question, and integrin blocking antibodies failed to have any effect. We conclude that integrins are involved in WNV infection but not at the level of binding to target cells.

Metabolic and immunomodulatory effects of n-3 fatty acids are different in mesenteric and epididymal adipose tissue of diet-induced obese mice.

In studies emphasizing antiobesogenic and anti-inflammatory effects of long-chain n-3 polyunsaturated fatty acids (LC-n-3 PUFA), diets with very high fat content, not well-defined fat quality, and extreme n-6/n-3 PUFA ratios have been applied frequently. Additionally, comparative analyses of visceral adipose tissues (VAT) were neglected. Considering the link of visceral obesity to insulin resistance or inflammatory bowel diseases, we hypothesized that VAT, especially mesenteric adipose tissue (MAT), may exhibit differential responsiveness to diets through modulation of metabolic and inflammatory processes. Here, we aimed to assess dietary LC-n-3 PUFA effects on MAT and epididymal adipose tissue (EAT) and on MAT-adjacent liver and intestine in diet-induced obese mice fed defined soybean/palm oil-based diets. High-fat (HF) and LC-n-3 PUFA-enriched high-fat diet (HF/n-3) contained moderately high fat with unbalanced and balanced n-6/n-3 PUFA ratios, respectively. Body composition/organ analyses, glucose tolerance test, measurements of insulin, lipids, mRNA and protein expression, and immunohistochemistry were applied. Compared with HF, HF/n-3 mice showed reduced fat mass, smaller adipocytes in MAT than EAT, improved insulin level, and lower hepatic triacylglycerol and plasma NEFA levels, consistent with liver and brown fat gene expression. Gene expression arrays pointed to immune cell activation in MAT and alleviation of intestinal endothelial cell activation. Validations demonstrated simultaneously upregulated pro- (TNFα, MCP-1) and anti-inflammatory (IL-10) cytokines and M1/M2-macrophage markers in VAT and reduced CD4/CD8α expression in MAT and spleen. Our data revealed differential responsiveness to diets for VAT through preferentially metabolic alterations in MAT and inflammatory processes in EAT. LC-n-3 PUFA effects were pro- and anti-inflammatory and disclose T cell-immunosuppressive potential.

Hepatic methionine homeostasis is conserved in C57BL/6N mice on high-fat diet despite major changes in hepatic one-carbon metabolism.

Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine ß-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.

Diet-induced obesity in ad libitum-fed mice: food texture overrides the effect of macronutrient composition.

Diet-induced obesity in mice can be achieved through the use of diets with different macronutrient compositions and textures. We aimed at determining the contribution of macronutrient composition to obesity development and associated pathophysiological changes in mice. C57BL/6N mice were offered a control, a high-fat or a Western-style diet, either as pellet (H for hard) or with identical composition in powder form (S for soft), resulting in C-S, C-H, HF-H, HF-S, W-H and W-S groups, respectively. Body fat distribution, expression levels of selected target genes in adipose tissues, clinical chemistry and hormone concentration in the blood, as well as liver TAG content were measured. The most striking finding was that all mice fed the different powder diets developed obesity with similar weight gain, whereas among the mice fed the pellet diets, only those given the HF and W diets became obese. This allowed us to separate diet-specific effects from obesity-mediated effects. Irrespective of the food texture, the W diet induced a more severe hepatosteatosis and higher activities of serum transaminases compared with the two other diets. Adipose tissue gene expression analysis revealed that leptin and adiponectin levels were not affected by the dietary composition per se, whereas uncoupling protein 1 and 11ß-hydroxysteroid dehydrogenase type 1 levels were decreased by both dietary composition and changes in body weight. In conclusion, diets differing in macronutrient composition elicit specific pathophysiological changes, independently of changes in body weight. A diet high in both fat and sugars seems to be more deleterious for the liver than a HF diet.

Effect of reducing the n-6:n-3 long-chain PUFA ratio during pregnancy and lactation on infant adipose tissue growth within the first year of life: an open-label randomized controlled trial.

BACKGROUND: The composition of long-chain PUFAs (LCPUFAs) in the maternal diet may affect obesity risk in the mother's offspring. OBJECTIVE: We hypothesized that a reduction in the n-6 (omega-6):n-3 (omega-3) LCPUFA ratio in the diet of pregnant women and breastfeeding mothers may prevent expansive adipose tissue growth in their infants during the first year of life. DESIGN: In a randomized controlled trial, 208 healthy pregnant women were randomly assigned to an intervention (1200 mg n-3 LCPUFAs as a supplement per day and a concomitant reduction in arachidonic acid intake) or a control diet from the 15th wk of pregnancy to 4 mo of lactation. The primary outcome was infant fat mass estimated by skinfold thickness (SFT) measurements at 4 body sites at 3-5 d, 6 wk, and 4 and 12 mo postpartum. Secondary endpoints included sonographic assessment of abdominal subcutaneous and preperitoneal fat, fat distribution, and child growth. RESULTS: Infants did not differ in the sum of their 4 SFTs at ≤1 y of life [intervention: 24.1 ± 4.4 mm (n = 85); control: 24.1 ± 4.1 mm (n = 80); mean difference: -0.0 mm (95% CI: -1.3, 1.3 mm)] or in growth. Likewise, longitudinal ultrasonography showed no significant differences in abdominal fat mass or fat distribution. CONCLUSIONS: We showed no evidence that supplementation with n-3 fatty acids and instructions to reduce arachidonic acid intake during pregnancy and lactation relevantly affects fat mass in offspring during the first year of life. Prospective long-term studies are needed to explore the efficacy of this dietary approach for primary prevention. This trial was registered at clinicaltrials.gov as NCT00362089.

Functional analyses reveal the greater potency of preadipocytes compared with adipocytes as endothelial cell activator under normoxia, hypoxia, and TNFalpha exposure.

Obesity is associated with a state of chronic low-grade inflammation. Immune cells accumulate in white adipose tissue (WAT). The vascular endothelium plays an interactive role in these infiltration and inflammatory processes. Mature and hypertrophic adipocytes are considered as the major adipogenic cell type secreting proinflammatory cytokines in WAT. In contrast, the proinflammatory capacity of preadipocytes and their role in endothelial cell activation have been neglected so far. To gain new insights into this molecular and cellular cross-talk, we examined the proinflammatory expression and secretion of normoxia, hypoxia, and TNFalpha-treated human preadipocytes and adipocytes (SGBS cells) and their impact on human microvascular endothelial cell (HMEC-1) function. In this study, stimulation of HMEC-1 with conditioned media (CM) from preadipocytes increased endothelial ICAM-1 expression and monocyte adhesion but not adipocyte-CM. After hypoxia and TNFalpha stimulation of SGBS cells, adipocyte-CM induced and preadipocyte-CM enhanced the monocyte adhesion. Concordantly, the expression of proinflammatory adipokines was considerably higher in preadipocytes than in adipocytes. SGBS-CM upregulated the phosphorylation of three MAPK pathways, STAT1/3, and c-Jun in HMEC-1, whereas the NF-kappaB pathway was not affected. Inhibitor experiments showed that monocyte/endothelial cell-cell adhesion and endothelial ICAM-1 expression was JNK and JAK-1/STAT1/3 pathway dependent and revealed IL-6 as a major mediator in CM increasing monocyte/endothelial cell-cell adhesion via the STAT1/3 pathway. Our study shows that preadipocytes rather than adipocytes operate as potent activators of endothelial cells. This can be enhanced in preadipocytes and induced in adipocytes by TNFalpha and hypoxia in a manner similar to what may occur in WAT in the etiology of obesity.

Rapid leukocyte migration by integrin-independent flowing and squeezing.

All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

Loss of nidogen-1 and -2 results in syndactyly and changes in limb development.

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice.

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.

A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth.

The goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell-surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology.

Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins.

Mouse embryos genetically null for the alphav integrin subunit develop intracerebral hemorrhages at midgestation and die shortly after birth. A key question is whether the hemorrhage arises from primary defects in vascular endothelial cells or pericytes or from other causes. We have previously reported normal initiation of cerebral vessels comprising branched tubes of endothelial cells. Here we show that the onset of hemorrhage is not due to defects in pericyte recruitment. Additionally, most alphav-null vessels display ultrastructurally normal endothelium-pericyte associations and normal interendothelial cell junctions. Thus, endothelial cells and pericytes appear to establish their normal relationships in cerebral microvessels. However, by both light and electron microscopy, we detected defective associations between cerebral microvessels and the surrounding brain parenchyma, composed of neuroepithelial cells, glia, and neuronal precursors. These data suggest a novel role for alphav integrins in the association between cerebral microvessels and central nervous system parenchymal cells.

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice.

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.