Myron E. Hinsdale

Apo E structure determines VLDL clearance and atherosclerosis risk in mice

We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice.

Limb, genital, CNS, and facial malformations result from gene/environment-induced cholesterol deficiency: Further evidence for a link to sonic hedgehog

Low cholesterol levels produced by treating cholesterol deficient mutant mice with a cholesterol synthesis inhibitor (BM 15.766) between days 4 to 7 of pregnancy resulted in malformations consistent with those in the Smith-Lemli-Opitz syndrome (SLOS). Facial anomalies in mildly affected gestational day 12 mouse embryos included a small nose and long upper lip; in more severely affected embryos, the facial and forebrain anomalies are representative of holoprosencephaly. Additionally, abnormalities of the mid- and hind-brain were observed and included stenosis of the cerebral aqueduct at the level of the isthmus and apparent absence of the organ progenitor for the cerebellar vermis. Although not previously directly linked to cholesterol deficiency in experimental animals, limb and external genital defects were a notable outcome in this multifactorially-based cholesterol deficiency model. The results of this study provide new evidence supporting an important role for cholesterol in early embryonic development, provide additional support for the hypothesis that this role may involve the function of specific gene products, such as sonic hedgehog (shh) signaling protein, and provide a description of the pathogenesis of some of the characteristic malformations in SLOS.

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.

Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects ≈500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

MicroRNA-26b Modulates the NF-κB Pathway in Alveolar Macrophages by Regulating PTEN

NF-κB is one of the best-characterized transcription factors, providing the link between early membrane–proximal signaling events and changes in many inflammatory genes. MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. In this study, we evaluated the role of miR-26b in the LPS-induced inflammatory response in bovine alveolar macrophages (bAMs). LPS stimulation of bAMs upregulated miR-26b at 1 h and downregulated it at 6 and 36 h. Overexpression of miR-26b in bAMs enhanced the LPS-induced mRNA expression of proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-8, and IL-10, but it directly inhibited that of IL-6. A similar trend was observed for the release of these cytokines and chemokines from bAMs. miR-26b directly bound the 3′-untranslated region of PTEN, leading to the reduction of PTEN protein in bAMs. miR-26b also enhanced the LPS-induced NF-κB signaling pathway, as revealed by increased NF-κB transcriptional activity and phosphorylation of p65, IκBα, IκB kinase, and Akt. Moreover, PTEN silencing increased the LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, IL-8, and IL-10 and upregulated the NF-κB pathway. Taken together, we conclude that miR-26b participates in the inflammatory response of LPS-stimulated bAMs by modulating the NF-κB pathway through targeting PTEN.

PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (VSMCs). However, the direct effects of PDGF receptor β (PDGFRβ) activation on VSMCs have not been studied in the context of atherosclerosis. Here, we present a new mouse model of atherosclerosis with an activating mutation in PDGFRβ. Increased PDGFRβ signaling induces chemokine secretion and leads to leukocyte accumulation in the adventitia and media of the aorta. Furthermore, PDGFRβD849V amplifies and accelerates atherosclerosis in hypercholesterolemic ApoE−/− or Ldlr−/− mice. Intriguingly, increased PDGFRβ signaling promotes advanced plaque formation at novel sites in the thoracic aorta and coronary arteries. However, deletion of the PDGFRβ-activated transcription factor STAT1 in VSMCs alleviates inflammation of the arterial wall and reduces plaque burden. These results demonstrate that PDGFRβ pathway activation has a profound effect on vascular disease and support the conclusion that inflammation in the outer arterial layers is a driving process for atherosclerosis.

Regulation of myofibroblast differentiation by miR-424 during epithelial-to-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is one of the most common and severe interstitial lung diseases. Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to IPF. MicroRNAs (miRNAs) are small non-coding RNAs of 18-24 nucleotides in length which regulate gene expression. Several studies have implicated miRNAs in EMT; however, specific miRNAs that regulate EMT in IPF have not yet been identified. In this study, we identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of α-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-β signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-β signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-β signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-β signaling pathway, likely through Smurf2.

Homozygosity for Frameshift Mutations in XYLT2 Result in a Spondylo-Ocular Syndrome with Bone Fragility, Cataracts, and Hearing Defects

Heparan and chondroitin/dermatan sulfated proteoglycans have a wide range of roles in cellular and tissue homeostasis including growth factor function, morphogen gradient formation, and co-receptor activity. Proteoglycan assembly initiates with a xylose monosaccharide covalently attached by either xylosyltransferase I or II. Three individuals from two families were found that exhibited similar phenotypes. The index case subjects were two brothers, individuals 1 and 2, who presented with osteoporosis, cataracts, sensorineural hearing loss, and mild learning defects. Whole exome sequence analyses showed that both individuals had a homozygous c.692dup mutation (GenBank: NM_022167.3) in the xylosyltransferase II locus (XYLT2) (MIM: 608125), causing reduced XYLT2 mRNA and low circulating xylosyltransferase (XylT) activity. In an unrelated boy (individual 3) from the second family, we noted low serum XylT activity. Sanger sequencing of XYLT2 in this individual revealed a c.520del mutation in exon 2 that resulted in a frameshift and premature stop codon (p.Ala174Profs(∗)35). Fibroblasts from individuals 1 and 2 showed a range of defects including reduced XylT activity, GAG incorporation of (35)SO4, and heparan sulfate proteoglycan assembly. These studies demonstrate that human XylT2 deficiency results in vertebral compression fractures, sensorineural hearing loss, eye defects, and heart defects, a phenotype that is similar to the autosomal-recessive disorder spondylo-ocular syndrome of unknown cause. This phenotype is different from what has been reported in individuals with other linker enzyme deficiencies. These studies illustrate that the cells of the lens, retina, heart muscle, inner ear, and bone are dependent on XylT2 for proteoglycan assembly in humans.

Regulation of the apolipoprotein B in heterozygous hypobetalipoproteinemic knock-out mice expressing truncated apoB, B81. Low production and enhanced clearance of apoB cause low levels of apoB

Low levels of cholesterol are protective against development of coronary artery disease. Heterozygous hypobetalipoproteinemic individuals expressing truncated apolipoprotein (apo)B as a result of mutation in the capob gene have low levels of cholesterol and apoB in their plasma. To study the molecular mechanism of low levels of apoB in these individuals, we employed a previously reported knock out mouse model generated by targeted modification of the apob gene. The heterozygous, apoB-100/B-81, mice express full length and truncated apoB, B-81, and have 20 and 35% lower levels of total cholesterol and apoB, respectively, when compared to WT (apoB-100/B-100) mice. The majority of the truncated apoB, B-81, fractionated in the VLDL- density range. The mechanism of low levels of apoB in B-100/B-81 mice was examined. Total hepatic apoB mRNA levels decreased by 15%, primarily due to lower levels of apoB-81 mRNA. Since apoB mRNA transcription rates were similar in B-100/B-100 and B-100/B-81 mice, low levels of mutant apoB-81 mRNA occurred by enhanced degradation of apoB mRNA transcript containing premature translational stop codon. ApoB synthesis measured on isolated hepatocytes decreased in B-100/B-81 mice by 35%, while apoB-48, apoE, and apoAI syntheses remained unchanged. Metabolic studies using whole animal showed a 32% decrease in triglyceride secretion rates, consistent with the apoB secretion rates. Inhibition of receptor-mediated clearance of apoB-81-containing particles resulted in greater relative accumulation of apoB-81 in plasma than apoB-100, suggesting enhanced clearance of apoB-81-containing particles. These results demonstrate that low levels of apoB in heterozygous hypobetalipoproteinemic mice occurs by low rates of apoB secretion, and increased clearance of truncated apoB. Similar mechanisms appear to contribute to low levels of apoB in hypobetalipoproteinemic humans.

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum

Circulating glycosyltransferases including xylosyltransferases I (XylT1) and II (XylT2) are potential serum biomarkers for various diseases. Understanding what influences the serum activity of these enzymes as well as the sources of these enzymes is important to interpreting the significance of alterations in enzyme activity during disease. This article demonstrates that in the mouse and human the predominant XylT in serum is XylT2. Furthermore, that total XylT levels in human serum are approximately 200% higher than those in plasma due in part to XylT released by platelets during blood clotting in vitro. In addition, the data from Xylt2 knock-out mice and mice with liver neoplasia show that liver is a significant source of serum XylT2 activity. The data presented suggest that serum XylT levels may be an informative biomarker in patients who suffer from diseases affecting platelet and/or liver homeostasis.