A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP's role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a "floxed" Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only ∼20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTPdeficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.J. Clin. Invest. 103:1287-1298(1999 to play an important role in this first "apo B lipidation" step (1,21,25). The existence of apo B in the rough ER of hepatocytes has been documented by immunoelectron microscopy (16). However, the lipidated apo B particles cannot be seen in the rough ER by routine electron microscopy, even when the thin sections are stained for lipids, because those particles are too small (< 150 Å in diameter) and lipid poor to be resolved by this technique. In a second step, the lipidated apo B molecule is thought to acquire the bulk of its core lipids by fusing with a large, VLDL-sized, apo B-free triglyceride particle (a "second-step" particle) (17,23). The existence of the secondstep triglyceride particles within a special compartment of the smooth ER has been supported by 2 different electron microscopic studies (16,23). Biochemical studies of VLDL assembly have also supported the concept that the bulk of neutral lipids are added to apo B in a second step after its translation is complete (1, 24).The role, if any, of MTP in the formation of second-step lipid particles is unclear. In fact, this issue has recently been highlighted as one of the fundamental problems in understanding MTP and lipoprotein assembly (15,26). In recent years, several groups have tried to address this issue by performing metabolic la...

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Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp؉͞؊ ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (Mttp؉͞؉), chow-fed Mttp؉͞؊ mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the Mttp؉͞؊ mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in Mttp؉͞؉ mice. Both the livers of adult Mttp؉͞؊ mice and the visceral endoderm of the yolk sacs from Mttp؉͞؊ embryos manifested an accumulation of cytosolic fat. All homozygous embryos (Mttp؊͞؊) died during embryonic development. In the visceral endoderm of Mttp؊͞؊ yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.Abetalipoproteinemia is an inherited human disease characterized by extremely low plasma levels of cholesterol and triglycerides and a virtual absence of the apolipoprotein (apo) B-containing lipoproteins [chylomicrons, very-low-density lipoproteins (VLDL), and low-density lipoproteins (LDL)] in the plasma (1, 2). Affected humans manifest intestinal fat malabsorption and frequently develop severe neurological problems as a result of deficient intestinal absorption of vitamin E, a fat-soluble vitamin (3). Abetalipoproteinemia is caused by mutations in the gene for the 97-kDa catalytic subunit of microsomal triglyceride transfer protein (MTP) (4-6). MTP is thought to transfer lipids to the apoB polypeptide chain as it is translated on the ribosome, allowing apoB to translocate into the lumen of the endoplasmic reticulum and assume the proper conformation for lipoprotein assembly (7,8). In abetalipoproteinemia, apoB is synthesized but cannot form lipoproteins and is degraded (9).Abetalipoproteinemia is considered to be an autosomal recessive syndrome, requiring two defective MTP alleles for disease expression. Parents of affected patients are said to have plasma lipid levels within the normal range (1). The normal plasma lipid levels in obligate heterozygotes have given rise to the concept that MTP normally is present in great excess within lipoprotein-secreting cells. That is, if MTP normally were present within microsomes in great excess, then halfnormal MTP levels in obligate heterozygotes would not be expected to affect lipoprotein secretion rates or plasma lipid levels. Against this concept, however, are recent in vitro data...