Apolipoprotein E receptor 2 deficiency decreases endothelial adhesion of monocytes and protects against autoimmune encephalomyelitis.

Under normal conditions, the blood-brain barrier effectively regulates the passage of immune cells into the central nervous system (CNS). However, under pathological conditions such as multiple sclerosis (MS), leukocytes, especially monocytes, infiltrate the CNS where they promote inflammatory demyelination, resulting in paralysis. Therapies targeting the immune cells directly and preventing leukocyte infiltration exist for MS but may compromise the immune system. Here, we explore how apolipoprotein E receptor 2 (ApoER2) regulates vascular adhesion and infiltration of monocytes during inflammation. We induced experimental autoimmune encephalitis in ApoER2 knockout mice and in mice carrying a loss-of-function mutation in the ApoER2 cytoplasmic domain. In both models, paralysis and neuroinflammation were largely abolished as a result of greatly diminished monocyte adherence due to reduced expression of adhesion molecules on the endothelial surface. Our findings expand our mechanistic understanding of the vascular barrier, the regulation of inflammation and vascular permeability, and the therapeutic potential of ApoER2-targeted therapies.

SORL1 deficiency in human excitatory neurons causes APP-dependent defects in the endolysosome-autophagy network.

Dysfunction of the endolysosomal-autophagy network is emerging as an important pathogenic process in Alzheimer's disease. Mutations in the sorting receptor-encoding gene SORL1 cause autosomal-dominant Alzheimer's disease, and SORL1 variants increase risk for late-onset AD. To understand the contribution of SORL1 mutations to AD pathogenesis, we analyze the effects of a SORL1 truncating mutation on SORL1 protein levels and endolysosome function in human neurons. We find that truncating mutation results in SORL1 haploinsufficiency and enlarged endosomes in human neurons. Analysis of isogenic SORL1 wild-type, heterozygous, and homozygous null neurons demonstrates that, whereas SORL1 haploinsufficiency results in endosome dysfunction, complete loss of SORL1 leads to additional defects in lysosome function and autophagy. Neuronal endolysosomal dysfunction caused by loss of SORL1 is relieved by extracellular antisense oligonucleotide-mediated reduction of APP protein, demonstrating that PSEN1, APP, and SORL1 act in a common pathway regulating the endolysosome system, which becomes dysfunctional in AD.

Angiotensin II Infusion Leads to Aortic Dissection in LRP8 Deficient Mice.

Myeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation. METHODS: LRP8+/+ and LRP8-/- mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining. RESULTS: AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8+/+ as well as LRP8-/- mice. However, only LRP8-/- mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8-/- mice. CONCLUSION: AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans.

VLDLR is not essential for reelin-induced neuronal aggregation but suppresses neuronal invasion into the marginal zone.

In the developing neocortex, radially migrating neurons stop migration and form layers beneath the marginal zone (MZ). Reelin plays essential roles in these processes via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). Although we recently reported that reelin causes neuronal aggregation via ApoER2, which is thought to be important for the subsequent layer formation, it remains unknown what effect reelin exerts via the VLDLR. Here, we found that ectopic reelin overexpression in the Vldlr-mutant mouse cortex causes neuronal aggregation, but without an MZ-like cell-sparse central region that is formed when reelin is overexpressed in the normal cortex. We also found that both the early-born and late-born Vldlr-deficient neurons invade the MZ and exhibit impaired dendrite outgrowth from before birth. Rescue experiments indicate that VLDLR suppresses neuronal invasion into the MZ via a cell-autonomous mechanism, possibly mediated by Rap1, integrin and Akt. These results suggest that VLDLR is not a prerequisite for reelin-induced neuronal aggregation and that the major role of VLDLR is to suppress neuronal invasion into the MZ during neocortical development.

Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice.

Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.

Gugulipid causes hypercholesterolemia leading to endothelial dysfunction, increased atherosclerosis, and premature death by ischemic heart disease in male mice.

For proper cholesterol metabolism, normal expression and function of scavenger receptor class B type I (SR-BI), a high-density lipoprotein (HDL) receptor, is required. Among the factors that regulate overall cholesterol homeostasis and HDL metabolism, the nuclear farnesoid X receptor plays an important role. Guggulsterone, a bioactive compound present in the natural product gugulipid, is an antagonist of this receptor. This natural product is widely used globally as a natural lipid-lowering agent, although its anti-atherogenic cardiovascular benefit in animal models or humans is unknown. The aim of this study was to determine the effects of gugulipid on cholesterol homeostasis and development of mild and severe atherosclerosis in male mice. For this purpose, we evaluated the impact of gugulipid treatment on liver histology, plasma lipoprotein cholesterol, endothelial function, and development of atherosclerosis and/or ischemic heart disease in wild-type mice; apolipoprotein E knockout mice, a model of atherosclerosis without ischemic complications; and SR-B1 knockout and atherogenic-diet-fed apolipoprotein E hypomorphic (SR-BI KO/ApoER61h/h) mice, a model of lethal ischemic heart disease due to severe atherosclerosis. Gugulipid administration was associated with histological abnormalities in liver, increased alanine aminotransferase levels, lower hepatic SR-BI content, hypercholesterolemia due to increased HDL cholesterol levels, endothelial dysfunction, enhanced atherosclerosis, and accelerated death in animals with severe ischemic heart disease. In conclusion, our data show important adverse effects of gugulipid intake on HDL metabolism and atherosclerosis in male mice, suggesting potential and unknown deleterious effects on cardiovascular health in humans. In addition, these findings reemphasize the need for rigorous preclinical and clinical studies to provide guidance on the consumption of natural products and regulation of their use in the general population.

Selenoprotein P and apolipoprotein E receptor-2 interact at the blood-brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration.

Selenoprotein P (Sepp1) and its receptor, apolipoprotein E receptor 2 (apoER2), account for brain retaining selenium better than other tissues. The primary sources of Sepp1 in plasma and brain are hepatocytes and astrocytes, respectively. ApoER2 is expressed in varying amounts by tissues; within the brain it is expressed primarily by neurons. Knockout of Sepp1 or apoER2 lowers brain selenium from ∼120 to ∼50 ng/g and leads to severe neurodegeneration and death in mild selenium deficiency. Interactions of Sepp1 and apoER2 that protect against this injury have not been characterized. We studied Sepp1, apoER2, and brain selenium in knockout mice. Immunocytochemistry showed that apoER2 mediates Sepp1 uptake at the blood-brain barrier. When Sepp1(-/-) or apoER2(-/-) mice developed severe neurodegeneration caused by mild selenium deficiency, brain selenium was ∼35 ng/g. In extreme selenium deficiency, however, brain selenium of ∼12 ng/g was tolerated when both Sepp1 and apoER2 were intact in the brain. These findings indicate that tandem Sepp1-apoER2 interactions supply selenium for maintenance of brain neurons. One interaction is at the blood-brain barrier, and the other is within the brain. We postulate that Sepp1 inside the blood-brain barrier is taken up by neurons via apoER2, concentrating brain selenium in them.

LRP8 mediates Wnt/ß-catenin signaling and controls osteoblast differentiation.

The Wnt/ß-catenin signaling pathway plays a pivotal role in regulating osteoblast differentiation and bone formation. Here, we identify low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) as a positive regulator of Wnt/ß-catenin signaling. In a small interfering RNA (siRNA) screen, LRP8 was shown to be required for Wnt/ß-catenin-induced transcriptional reporter activity. We found that ectopic expression of LRP8 increased Wnt-induced transcriptional responses, and promoted Wnt-induced ß-catenin accumulation. Moreover, knockdown of LRP8 resulted in a decrease in ß-catenin levels and suppression of Wnt/ß-catenin-induced Axin2 transcription. Functional studies in KS483 osteoprogenitor cells showed that LRP8 depletion resulted in impaired activation of endogenous Wnt-induced genes and decreased osteoblast differentiation and mineralization, whereas LRP8 ectopic expression had the opposite effect. These results identify LRP8 as a novel positive factor of canonical Wnt signaling pathway and show its involvement in Wnt-induced osteoblast differentiation.

ApoE receptor 2 regulates synapse and dendritic spine formation.

BACKGROUND: Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation-processes critical for learning and memory. METHODOLOGY/PRINCIPAL FINDINGS: In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density. CONCLUSIONS/SIGNIFICANCE: These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.

Apolipoprotein E induces antiinflammatory phenotype in macrophages.

OBJECTIVE: Apolipoprotein E (apoE) exerts potent antiinflammatory effects. Here, we investigated the effect of apoE on the functional phenotype of macrophages. METHODS AND RESULTS: Human apoE receptors very-low-density lipoprotein receptor (VLDL-R) and apoE receptor-2 (apoER2) were stably expressed in RAW264.7 mouse macrophages. In these cells, apoE downregulated markers of the proinflammatory M1 phenotype (inducible nitric oxide synthase, interleukin [IL]-12, macrophage inflammatory protein-1α) but upregulated markers of the antiinflammatory M2 phenotype (arginase I, SOCS3, IL-1 receptor antagonist [IL-1RA]). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis), as well as poly(I:C)- or interferon-γ-induced production of proinflammatory cytokines; cyclooxygenase-2 expression; and activation of nuclear factor-κB, IκB, and STAT1, were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of the M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice but not wild-type or low-density lipoprotein receptor-deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38 mitogen-activated protein kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38 mitogen-activated protein kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE-/- mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels, and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression). CONCLUSIONS: ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the proinflammatory M1 to the antiinflammatory M2 phenotype. This effect may represent a novel antiinflammatory activity of apoE.

Antiphospholipid antibodies promote leukocyte-endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via ß2GPI and apoER2.

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to ß2 glycoprotein I (ß2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of ß2GPI and ß2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by ß2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Functional interactions between the LRP6 WNT co-receptor and folate supplementation.

Crooked tail (Cd) mice bear a gain-of-function mutation in Lrp6, a co-receptor for canonical WNT signaling, and are a model of neural tube defects (NTDs), preventable with dietary folic acid (FA) supplementation. Whether the FA response reflects a direct influence of FA on LRP6 function was tested with prenatal supplementation in LRP6-deficient embryos. The enriched FA (10 ppm) diet reduced the occurrence of birth defects among all litters compared with the control (2 ppm FA) diet, but did so by increasing early lethality of Lrp6(-/-) embryos while actually increasing NTDs among nulls alive at embryonic days 10-13 (E10-13). Proliferation in cranial neural folds was reduced in homozygous Lrp6(-/-) mutants versus wild-type embryos at E10, and FA supplementation increased proliferation in wild-type but not mutant neuroepithelia. Canonical WNT activity was reduced in LRP6-deficient midbrain-hindbrain at E9.5, demonstrated in vivo by a TCF/LEF-reporter transgene. FA levels in media modulated the canonical WNT response in NIH3T3 cells, suggesting that although FA was required for optimal WNT signaling, even modest FA elevations attenuated LRP5/6-dependent canonical WNT responses. Gene expression analysis in embryos and adults showed striking interactions between targeted Lrp6 deficiency and FA supplementation, especially for mitochondrial function, folate and methionine metabolism, WNT signaling and cytoskeletal regulation that together implicate relevant signaling and metabolic pathways supporting cell proliferation, morphology and differentiation. We propose that FA supplementation rescues Lrp6(Cd/Cd) fetuses by normalizing hyperactive WNT activity, whereas in LRP6-deficient embryos, added FA further attenuates reduced WNT activity, thereby compromising development.

Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.

Generation of Lrp6 conditional gene-targeting mouse line for modeling and dissecting multiple birth defects/congenital anomalies.

Lrp6 is a key coreceptor in the canonical Wnt pathway that is widely involved in tissue/organ morphogenesis. We generated a loxP-floxed Lrp6 mouse line. Crossing with a general Cre deleter, we obtained the Lrp6-floxdel mice, in which the loxP-floxed exon 2 of Lrp6 gene has been deleted ubiquitously. The homozygotes of Lrp6-floxdel mice reproduced typical defects as seen in the conventional Lrp6-deficient mice, such as defects in eye, limb, and neural tube, and die around birth. We also found new phenotypes including cleft palate and agenesis of external genitalia in the Lrp6-floxdel mice. In addition, the Lrp6-deficient embryos are known to be defective in other systems and internal organs including the heart and brain. Thus, by selectively crossing with a lineage-specific or inducible Cre mouse line, the Lrp6 conditional gene-targeting mice will allow us to model specific types of birth defects for mechanism and prevention studies.

Wnt/beta-catenin signaling blockade promotes neuronal induction and dopaminergic differentiation in embryonic stem cells.

Embryonic stem cells (ESCs) represent not only a promising source of cells for cell replacement therapy, but also a tool to study the molecular mechanisms underlying cellular signaling and dopaminergic (DA) neuron development. One of the main regulators of DA neuron development is Wnt signaling. Here we used mouse ESCs (mESCs) lacking Wnt1 or the low-density lipoprotein receptor-related protein 6 (LRP6) to decipher the action of Wnt/beta-catenin signaling on DA neuron development in mESCs. We provide evidence that the absence of LRP6 abrogates responsiveness of mESCs to Wnt ligand stimulation. Using two differentiation protocols, we show that the loss of Wnt1 or LRP6 increases neuroectodermal differentiation and the number of mESC-derived DA neurons. These effects were similar to those observed following treatment of mESCs with the Wnt/beta-catenin pathway inhibitor Dickkopf1 (Dkk1). Combined, our results show that decreases in Wnt/beta-catenin signaling enhance neuronal and DA differentiation of mESCs. These findings suggest that: 1) Wnt1 or LRP6 are not strictly required for the DA differentiation of mESCs in vitro, 2) the levels of morphogens and their activity in ESC cultures need to be optimized to improve DA differentiation, and 3) by enhancing the differentiation and number of ESC-derived DA neurons with Dkk1, the application of ESCs for cell replacement therapy in Parkinson's disease may be improved.

Wnt signaling as a therapeutic target for bone diseases.

BACKGROUND: There is a need to develop new bone anabolic agents because current bone regeneration regimens have limitations. The Wingless-type MMTV integration site (Wnt) pathway has emerged as a regulator of bone formation and regeneration. OBJECTIVE: To review the molecular basis for Wnt pathway modulation and discuss strategies that target it and improve bone mass. METHODS: Data in peer-reviewed reports and meeting abstracts are discussed. RESULTS/CONCLUSIONS: Neutralizing inhibitors of Wnt signaling have emerged as promising strategies. Small-molecule inhibitors of glycogen synthase kinase 3beta increase bone mass, lower adiposity and reduce fracture risk. Neutralizing antibodies to Dickkopf 1, secreted Frizzled-related protein 1 and sclerostin produce similar outcomes in animal models. These drugs are exciting breakthroughs but are not without risks. The challenges include tissue-specific targeting and consequently, long-term safety.

Mutation in EGFP domain of LDL receptor-related protein 6 impairs cellular LDL clearance.

Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6(R611C)) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known. Cholesterol uptake was examined in cells from LRP6(+/-) mice and LRP6(R611C) mutation carriers. Splenic B cells of LRP6(+/-) mice have significantly lower LRP6 expression and low-density lipoprotein (LDL) uptake than those of the wild-type littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6(R611C) mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wild-type receptors show similar affinities for apolipoprotein B at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1 (Niemann-Pick disease type C1), an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6(R611C) was lower compared to wild-type receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apolipoprotein B dissociation from LRP6(R611C) compared to wild-type receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes, which compromises their recycling to the plasma membrane.

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis.

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2(Tg)). The predicted protein encoded by Rspo2(Tg) neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2(Tg/Tg) mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2(Tg/Tg) lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2(Tg) and Lrp6(-) alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.