A catalog of associations between rare coding variants and COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19), a respiratory illness that can result in hospitalization or death. We investigated associations between rare genetic variants and seven COVID-19 outcomes in 543,213 individuals, including 8,248 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome-wide or when specifically focusing on (i) 14 interferon pathway genes in which rare deleterious variants have been reported in severe COVID-19 patients; (ii) 167 genes located in COVID-19 GWAS risk loci; or (iii) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, with results publicly browsable at https://rgc-covid19.regeneron.com.

Genome-wide analysis in 756,646 individuals provides first genetic evidence that ACE2 expression influences COVID-19 risk and yields genetic risk scores predictive of severe disease.

SARS-CoV-2 enters host cells by binding angiotensin-converting enzyme 2 (ACE2). Through a genome-wide association study, we show that a rare variant (MAF = 0.3%, odds ratio 0.60, P=4.5×10-13) that down-regulates ACE2 expression reduces risk of COVID-19 disease, providing human genetics support for the hypothesis that ACE2 levels influence COVID-19 risk. Further, we show that common genetic variants define a risk score that predicts severe disease among COVID-19 cases.

Predictive value of genomic screening: cross-sectional study of cystic fibrosis in 50,788 electronic health records.

Doubts have been raised about the value of DNA-based screening for low-prevalence monogenic conditions following reports of testing this approach using available electronic health record (EHR) as the sole phenotyping source. We hypothesized that a better model for EHR-focused examination of DNA-based screening is Cystic Fibrosis (CF) since the diagnosis is proactively sought within the healthcare system. We reviewed CFTR variants in 50,778 exomes. In 24 cases with bi-allelic pathogenic CFTR variants, there were 21 true-positives. We considered three cases "potential" false-positives due to limitations in available EHR phenotype data. This genomic screening exhibited a positive predictive value of 87.5%, negative predictive value of 99.9%, sensitivity of 95.5%, and a specificity of 99.9%. Despite EHR-based phenotyping limitations in three cases, the presence or absence of pathogenic CFTR variants has strong predictive value for CF diagnosis when EHR data is used as the sole phenotyping source. Accurate ascertainment of the predictive value of DNA-based screening requires condition-specific phenotyping beyond available EHR data.

A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

Gene expression profiling in subcutaneous, visceral and epigastric adipose tissues of patients with extreme obesity.

OBJECTIVE: The goal of the present study was to identify differences in gene expression between SAT, VAT and EAT depots in Class III severely obese individuals. DESIGN: Human subcutaneous (SAT) and visceral (VAT) adipose tissues exhibit differential gene expression profiles. There is little information, however, about the other proximal white adipose tissue, epigastric (EAT), in terms of its function and contribution to metabolism. SUBJECTS AND METHODS: Using RNA from adipose biospecimens obtained from Class III severely obese patients undergoing open Roux-en-Y gastric bypass surgery, we compared gene expression profiles between SAT, VAT and EAT, using microarrays validated by real-time quantitative PCR. RESULTS: The three depots were found to share 1907 genes. VAT had the greatest number of genes (66) expressed exclusively in this depot, followed by SAT (23), and then EAT (14). Moreover, VAT shared more genes with EAT (65) than with SAT (38). Further analyses using ratios of SAT/EAT, VAT/EAT and SAT/VAT identified specific as well as overlapping networks and pathways of genes representing dermatological diseases, inflammation, cell cycle and growth, cancer and development. Targeted analysis of genes, having a role in adipose tissue development and function, revealed that Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC1-α) that regulates the precursor of the hormone Irisin (FNCD5) were abundantly expressed in all three fat depots, along with fibroblast growth factors (FGF) FGF1, FGF7 and FGF10, whereas, FGF19 and FGF21 were undetectable. CONCLUSIONS: These data indicate that EAT has more in common with VAT, suggesting similar metabolic potential. The human epigastric adipose depot could have a significant functional role in metabolic diseases and should be further investigated.

Schwann cell type V collagen inhibits axonal outgrowth and promotes Schwann cell migration via distinct adhesive activities of the collagen and noncollagen domains.

Previously, we reported the cloning of alpha4 type V collagen, a novel member of the collagen type V gene family that is expressed by Schwann cells in developing peripheral nerves (Chernousov et al., 2000). The present study was performed to investigate the effects of this collagen on the adhesion and migration of premyelinating Schwann cells and neurite outgrowth from embryonic dorsal root ganglion neurons. Purified alpha4(V)-containing collagen isolated from Schwann cell conditioned medium (collagen type V(SC)) promoted migration of Schwann cells but inhibited outgrowth of axons from rat embryo dorsal root ganglia. Collagen type V(SC) blocked axonal outgrowth in the presence of otherwise active substrates such as collagen type IV, indicative of active inhibition. The noncollagen N-terminal domain of alpha4(V) promoted Schwann cell adhesion, spreading, and migration. These processes were inhibited by soluble heparin but not by function-blocking antibodies against alpha1- and alpha2-integrins. The collagen domain of pepsin-digested collagen type V was poorly adhesive for Schwann cells. The type V collagen domain but not the alpha4(V) N-terminal domain blocked neurite outgrowth from dorsal root ganglion neurons. In cocultures of dorsal root ganglion neurons and Schwann cells, collagen type V(SC) promoted axon fasciculation and association of axons with Schwann cells. These results suggest that in embryonic peripheral nerves, collagen type V(SC) plays a dual role in regulating cell migration. This represents a heretofore unrecognized function of peripheral nerve collagen fibrils in regulating patterns of peripheral nerve growth during development.

Elastase is not sufficient to induce experimental abdominal aortic aneurysms.

PURPOSE: Research investigating abdominal aortic aneurysms (AAAs) commonly uses a rat model dependent on aortic infusion of porcine pancreatic elastase to initiate AAA formation. Unfortunately, the sizes of AAAs generated by this model have varied widely among published studies. This may reflect lot-to-lot variations in commercial elastase preparations. This study was undertaken to investigate the ability of different lots of elastase to induce AAAs and explain the variability identified. METHODS: Four lots of elastase were evaluated in the standard rat AAA model. Saline solution was used as a control. Additional groups of rats were treated with higher concentrations of elastase with or without the macrophage activator thioglycollate medium. Aortic diameters were measured in all rats. Inflammation and elastin degradation was examined histologically. Elastase activity and purity were evaluated for all lots. RESULTS: Of the four lots tested, only one was able to consistently generate AAAs at the standard dose (P <.05). Increasing the amount of elastase infused produced AAAs in some ineffective lots. Infusion of thioglycollate medium in combination with otherwise ineffective elastase produced AAAs (P =.02). However, the elastase with the highest purity failed to generate AAAs, even at the highest dose tested or in combination with thyioglycollate medium. Thioglycollate medium alone failed to result in AAA formation. All elastase lots displayed elastolytic activity in vitro and produced elastin degradation in vivo. Elastin degradation did not correlate with AAA size in elastase-treated rats (P = NS). Aneurysm size correlated with extent of inflammation (P =.005). CONCLUSION: Induction of AAAs does not correlate with elastolytic activity. Infusion of pure elastase alone is not sufficient to induce AAA formation in spite of evidence of elastin degradation. Presumed inflammatory modifiers, which contaminate some elastase preparations, enhance AAA formation. Future use of this rat model will need to take the variability of elastase preparations into account with controls for each new elastase lot.

Inhibition of inducible nitric oxide synthase limits nitric oxide production and experimental aneurysm expansion.

PURPOSE: Nitric oxide (NO), frequently cited for its protective role, can also generate toxic metabolites known to degrade elastin. Both abdominal aortic aneurysms (AAAs) and inducible nitric oxide synthase (iNOS) are associated with inflammatory states, yet the relationship between NO production by iNOS and AAA development is unknown. The current study examines iNOS expression, NO production, and the effects of selective inhibition of iNOS by aminoguanidine in experimental AAA. METHODS: An intra-aortic elastase infusion model was used. Control rats received intra-aortic saline infusion and postoperative intraperitoneal saline injections (Group 1). In the remaining groups, intra-aortic elastase infusion was used to induce aneurysm formation. These rats were treated with intraperitoneal injections of saline postoperatively (Group 2), aminoguanidine postoperatively (Group 3), or aminoguanidine preoperatively and postoperatively (Group 4). Aortic diameter and plasma nitrite/nitrate levels were measured on the day of surgery and postoperative day 7. Aortas were harvested for biochemical and histologic analysis on postoperative day 7. RESULTS: Infusion of elastase produced AAAs (P <.001) with significant production of iNOS (P <.05) and nitrite/nitrate (P <.003) compared with controls. Selective inhibition of iNOS with aminoguanidine in elastase-infused aortas significantly reduced aneurysm size (P <.01) compared with elastase infusion alone. Aminoguanidine-treated rats displayed suppression of iNOS expression and plasma nitrite/nitrate production not significantly different from the control group. Histologic evaluation revealed equivalent inflammatory infiltrates in elastase-infused groups. CONCLUSION: Expression of iNOS is induced and plasma nitrite/nitrate levels are increased in experimental AAA. Inhibition of iNOS limits NO production and iNOS expression, resulting in smaller aneurysm size. NO production by iNOS plays an important role with detrimental effects during experimental aneurysm development.

In vivo regulation of syndecan-3 expression in the rat uterus by 17 beta-estradiol.

The immature rat uterus has been extensively used as an in vivo model system to study the molecular mechanisms of steroid hormone actions. In this study, we demonstrated the regulated expression of syndecan-3 in the rat uterus by the steroid hormone 17 beta-estradiol. Administration of a single physiological dose of 17 beta-estradiol (40 microg/kg) to ovariectomized immature animals induced a rapid and transient increase in uterine syndecan-3 mRNA. Transcript levels reached a peak elevation of 3-fold above saline control tissues 4 h after hormone administration. Inhibition of message up-regulation by actinomycin D but not cycloheximide indicated a hormone response dependent on RNA transcription but not new protein synthesis. The estrogenic ligands estriol and tamoxifen were also effective at raising syndecan-3 mRNA levels; however, nonestrogenic ligands, including progesterone, 5 alpha-dihydrotestosterone, and dexamethasone, failed to stimulate a change in mRNA levels. Hormone-induced changes in mRNA led to transient changes in syndecan-3 protein content and significant alteration in the temporal and spatial expression in endometrial epithelial cells. Collectively, these data show that the steroid hormone 17 beta-estradiol, regulates transcription of the syndecan-3 gene in the uterus via an estrogen receptor-dependent mechanism. This estrogen-regulated expression of syndecan-3 may play an important role in changes in tissue ultrastructure crucial for proper uterine growth.

Schwann cells synthesize type V collagen that contains a novel alpha 4 chain. Molecular cloning, biochemical characterization, and high affinity heparin binding of alpha 4(V) collagen.

Previously, we reported the isolation of a heparan sulfate-binding collagenous protein, p200, that is expressed by Schwann cells in developing peripheral nerves ((1996) J. Biol. Chem. 271, 13844-13853; (1999) J. Neurosci. Res. 56, 284-294). Here, we report the cloning of p200 cDNA from a Schwann cell cDNA library. The deduced amino acid sequence identifies p200 as a novel member of the collagen type V gene family. This polypeptide, which we have named alpha4 type V (alpha4(V)) collagen, contains an uninterrupted Gly-X-X collagen domain of 1011 amino acids that shows 82% sequence identity to human alpha3(V) collagen and 71% identity to rat alpha1(V) collagen. alpha4(V) is secreted by Schwann cells as a collagen heterotrimer that also contains alpha1(V) chains. alpha4(V)-containing collagen molecules synthesized by Schwann cells retain their amino-terminal non-collagenous domains. alpha4(V) mRNA was detected by reverse transcriptase-linked polymerase chain reaction amplification in neonatal and adult brain and neonatal peripheral nerve. alpha4(V) mRNA and protein were not detected in most other tissues, including the placenta and heart, which are known to contain alpha3(V). This pattern of alpha4(V) expression contrasted with that of alpha1(V) mRNA and protein, which were ubiquitously expressed. The isolated alpha4(V) chain demonstrated an unusually high affinity for heparin. The restricted expression and unusual properties of alpha4(V)-containing collagen type V molecules suggest a unique and important role for these molecules in peripheral nerve development.

Schwann cell extracellular matrix molecules and their receptors.

The major cellular constituents of the mammalian peripheral nervous system are neurons (axons) and Schwann cells. During peripheral nerve development Schwann cells actively deposit extracellular matrix (ECM), comprised of basal lamina sheets that surround individual axon-Schwann cell units and collagen fibrils. These ECM structures are formed from a diverse set of macromolecules, consisting of glyco-proteins, collagens and proteoglycans. To interact with ECM, Schwann cells express a number of integrin and non-integrin cell surface receptors. The expression of many Schwann cell ECM proteins and their receptors is developmentally regulated and, in some cases, dependent on axonal contact. Schwann cell ECM acts as an organizer of peripheral nerve tissue and strongly influences Schwann cell adhesion, growth and differentiation and regulates axonal growth during development and regeneration.

Antisense inhibition of syndecan-3 expression during skeletal muscle differentiation accelerates myogenesis through a basic fibroblast growth factor-dependent mechanism.

Syndecan-3 is a member of a family of transmembrane proteoglycans that posses highly homologous cytoplasmic and transmembrane domains and function as extracellular matrix receptors and low-affinity receptors for signaling molecules such as basic fibroblasts growth factor (FGF-2). Syndecan-3 is transiently expressed in developing limb bud and absent in adult skeletal muscle. In this study we investigated the expression of syndecan-3 and its role on FGF-2-dependent inhibition of myogenesis. Syndecan-3 expression was down-regulated during skeletal muscle differentiation of C(2)C(12) myoblasts, as determined by Northern blot analyses and immunoprecipitation. To probe the function of syndecan-3 during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid coding for antisense syndecan-3 mRNA. The resulting inhibition of syndecan-3 expression caused accelerated skeletal muscle differentiation, as determined by expression of creatine kinase and myosin and myoblast fusion. Expression of a master transcription factor for muscle differentiation, myogenin, was also accelerated in antisense syndecan-3-transfected myoblasts compared with control transfected and wild type cells. Reduced expression of syndecan-3 resulted in a 13-fold decrease in sensitivity to FGF-2-dependent inhibition of myogenin expression. Addition of heparin partially reversed this effect. These results demonstrate that syndecan-3 expression is down-regulated during differentiation and the level of expression of membrane-bound heparan sulfate on myoblast surface is critical for fine modulation of responsiveness to FGF-2. These findings strongly suggest a role for syndecan-3 in regulation of skeletal muscle terminal differentiation.

p200, a collagen secreted by Schwann cells, is expressed in developing nerves and in adult nerves following axotomy.

Previously we reported that cultured rat Schwann cells secrete p200, a collagen-like heparin-binding adhesive glycoprotein with a restricted pattern of expression. Here we report that p200 is secreted as a stable trimer, but only after treatment of Schwann cells with ascorbic acid, and was deposited in the fibrillar extracellular matrix. Heparin and heparitinase treatment inhibited incorporation of p200 into extracellular matrix, suggesting the involvement of Schwann cell heparan sulfate proteoglycans in this process. Pepsin digestion revealed that p200 secreted by ascorbate-treated cells contains a collagenous domain of approximately 140 kDa. Immunofluorescent staining of rat embryos at different ages showed that p200 first appeared between embryonic days 15 and 18, and was confined to peripheral nerves. Staining of adult peripheral nerve was negative, but p200 expression was induced in adult sciatic nerve following nerve transection. These data suggest that p200 carries out unique functions during peripheral nerve development and regeneration and that its expression by Schwann cells is regulated by axon-Schwann cell interaction.

The matrix metalloproteinase inhibitor BB-94 limits expansion of experimental abdominal aortic aneurysms.

PURPOSE: Matrix metalloproteinases (MMPs) are proteolytic enzymes that can degrade the extracellular matrix of the aortic wall and lead to the formation of abdominal aortic aneurysms (AAAs). MMP inhibitors are a class of drugs that were developed to inhibit the activity of these proteolytic enzymes and are currently being studied as a way to control inflammatory diseases and cancer metastases. In this project, BB-94 (also known as batimastat), a specific inhibitor of MMPs, was evaluated for its ability to control aneurysmal growth in an experimental AAA model. METHODS: Experimental AAAs were created in a standard rat model by perfusing elastase into an isolated segment of aorta. The rats then were randomized to postoperatively undergo treatment daily with the MMP inhibitor BB-94 or the carrier control solution. Measurements of the aortic diameter were made at the time of initial surgery and at the time of death on postoperative day 7. Aortic tissue was obtained for histologic examination, elastin evaluation, and MAC 1-alpha antibody staining to evaluate the inflammatory response. RESULTS: The rats that underwent treatment with BB-94 had significantly less aneurysmal dilatation and a 113% increase in aortic size, as compared with the control rats that had a 157% increase (P =.026). Histologic examination of the harvested aortas and grading of the elastin content showed a significantly greater elastin preservation in those rats that were treated with BB-94 as compared with the control rats (P =.036). MAC 1-alpha antibody staining showed an attenuation of the inflammatory response in the group of rats that underwent treatment with BB-94. Morphologic examination also revealed that the control of the inflammatory response correlated with the areas of elastin preservation. CONCLUSION: MMP inhibition with BB-94 limited the expansion of AAAs in this rat model. BB-94 appears to work not only as a direct pharmacologic inhibitor of MMPs but also as an interference with the inflammatory response seen in AAAs. Control of the inflammatory response was an unexpected result and may be related to the alterations in feedback mechanisms that are related to extracellular matrix degradation. Because this class of drugs is presently being developed to control the MMP inflammatory response seen with arthritis, these drugs also may ultimately serve as a pharmacologic treatment for patients with AAAs.

Syndecan-1 expression inhibits myoblast differentiation through a basic fibroblast growth factor-dependent mechanism.

Expression of syndecan-1, a cell-surface heparan sulfate proteoglycan, is down-regulated during skeletal muscle differentiation (Larraín, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J., and Brandan, E. (1997) J. Biol. Chem. 272, 18418-18424). We examined the role of syndecan-1 in basic fibroblast growth factor (bFGF)-dependent inhibition of myogenesis. C2C12 myoblasts were stably transfected with an expression plasmid containing the rat syndecan-1 coding region cDNA. Constitutive syndecan-1 expression resulted in a strongly diminished capacity of the transfected clones to differentiate and to express skeletal muscle-specific markers such as fusion, creatine kinase, and myosin. The expression of myogenin, a master transcription factor for muscle differentiation, was also reduced and delayed. Analysis of the induction of a myogenin promoter-driven reporter revealed that syndecan-1 expression resulted in a 6-7-fold increase in sensitivity to bFGF-dependent inhibition of myogenin expression. Transfecting the cells with a plasmid containing myogenin cDNA reversed the inhibition of myogenin transcriptional activation and myosin expression in syndecan-1-transfected cells; however, cell fusion was not observed. These results demonstrate that syndecan-1 expression enhances cell responsiveness to bFGF and inhibits myoblast fusion and suggest that muscle terminal differentiation is regulated by syndecan-1 expression.

Sensory and motor neuron-derived factor is a transmembrane heregulin that is expressed on the plasma membrane with the active domain exposed to the extracellular environment.

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Synergistic regulation of Schwann cell proliferation by heregulin and forskolin.

A peptide corresponding to the epidermal growth factor homology domain of beta-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95(RSK2), was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly.

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. alpha5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

Expression of matrix metalloproteinases and TIMPs in human abdominal aortic aneurysms.

Degradation of extracellular matrix, especially elastin, within the aortic wall is a hallmark of abdominal aortic aneurysms (AAAs). Normal turnover of matrix proteins is mediated by a family of enzymes called matrix metalloproteinases (MMPs). MMP activity is regulated by proteins called tissue inhibitors of metalloproteinases (TIMPs). We analyzed the expression of all known MMPs with established elastolytic activity and TIMPs in human AAA and control tissue. mRNA coding for MMP-9, MMP-2, human macrophage metalloelastase, MMP-7, TIMP-1, and TIMP-2 were amplified by reverse transcriptase-PCR in control and AAA tissue. A Northern blot assay was used to measure the levels of mRNA coding for MMP-2, MMP-9, TIMP-1, and TIMP-2. Control aortic tissue was obtained from patients with occlusive disease and from organ donors. The expression of MMP-7 and human macrophage metalloelastase was not detected in any aortic specimens. By Northern blot analysis the mean level of MMP-2 mRNA was not significantly different between control groups and AAAs (normalized values: occlusive, 1.5 +/- 0.8, n = 3; donor, 4.5 +/- 2.2, n = 6; AAA, 4.0 +/- 0.95, n = 15). There was a significant increase in the level of MMP-9 mRNA in AAA specimens (occlusive, 16.8 +/- 3, n = 3; donor, 5.7 +/- 1.2, n = 6; AAA, 56.7 +/- 11, n = 15, p = 0.0069). The levels of mRNA coding for TIMP-1 were not significantly different. There was a small but statistically significant increase in TIMP-2 mRNA in AAA tissue. These data support the hypothesis that increased activity of MMP-9, but not MMP-2, is an important factor in the etiology of AAAs. This enhanced MMP-9 activity could then result in degradation of the ECM, leading to aneurysmal dilatation.

Syndecan-1 is up-regulated in ras-transformed intestinal epithelial cells.

The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development. Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.