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Biomedical subjects

Karl Tryggvason

Publications and source records attributed to Karl Tryggvason.

At least 37 records · Page 2Linked to original sources
PubMed2005

Impeded interaction between Schwann cells and axons in the absence of laminin alpha4.

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Animals↗
PubMed2005

Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation.

Membrane type 1-matrix metalloprotease (MT1-MMP or MMP-14) is a major activator of pro-MMP-2 and is essential for skeletal development. We show here that it is required for branching morphogenesis of the submandibular gland but not the lung. Instead, in the lung, it is essential for postnatal development of alveolar septae. Lung development in Mmp14-/- mice is arrested at the prealveolar stage with compensatory hyperinflation of immature saccules. Mmp2-/- mice lacked comparable defects in the lung and submandibular gland, suggesting that MT1-MMP acts via mechanisms independent of pro-MMP-2 activation. Since the developmental defects in the lung are first manifest around the time of initial vascularization (E16.5), we investigated the behavior of pulmonary endothelial cells from Mmp14+/+ and Mmp14-/- mice. Endothelial cells from lungs of 1-week-old Mmp14-/- mice show reduced migration and formation of three-dimensional structures on Matrigel. Since pulmonary septal development requires capillary growth, the underlying mechanism of pulmonary hypoplasia in Mmp14-/- mice may be defective angiogenesis, supporting a model in which angiogenesis is a critical rate-limiting step for acquisition of pulmonary parenchymal mass.

Animals↗
PubMed2005

A simplified laminin nomenclature.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Animals↗
PubMed2005

Characterization of the interactions of the nephrin intracellular domain.

Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

Amino Acid Sequence↗
PubMed2005

How does the kidney filter plasma?

The kidneys filter the plasma in special filtration units-glomeruli-and thereby excrete low-molecular-weight waste products into the urine. The mechanisms of glomerular filtration have been a matter of controversy for several decades, but recent data have revealed new details about the molecular nature of the filter and have demonstrated a central role for the podocyte slit diaphragm in the filtration process.

Animals↗
PubMed2004

Membrane type 1-matrix metalloproteinase is regulated by chemokines monocyte-chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in endothelial cells during angiogenesis.

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.

Actins↗
PubMed2004

The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles.

Alveolar macrophages (AMs) express the class A scavenger receptor macrophage receptor with collagenous structure (MARCO), but its role in vivo in lung defense against bacteria and environmental particles has not been studied. We used MARCO-deficient mice to directly test the in vivo role of AM MARCO in innate defense against pneumococcal infection and environmental particles. In a murine model of pneumococcal pneumonia, MARCO(-/-) mice displayed an impaired ability to clear bacteria from the lungs, increased pulmonary inflammation and cytokine release, and diminished survival. In vitro binding of Streptococcus pneumoniae and in vivo uptake of unopsonized particles by MARCO(-/-) AMs were dramatically impaired. MARCO(-/-) mice treated with the "inert" environmental particle TiO(2) showed enhanced inflammation and chemokine expression, indicating that MARCO-mediated clearance of inert particles by AMs prevents inflammatory responses otherwise initiated by other lung cells. Our findings point to an important role of MARCO in mounting an efficient and appropriately regulated innate immune response against inhaled particles and airborne pathogens.

Animals↗
PubMed2004

Impaired angiogenesis, delayed wound healing and retarded tumor growth in perlecan heparan sulfate-deficient mice.

Perlecan, a modular proteoglycan carrying primary heparan sulfate (HS) side chains, is a major component of blood vessel basement membranes. It sequesters growth factors such as fibroblast growth factor 2 (FGF-2) and regulates the ligand-receptor interactions on the cell surface, and thus it has been implicated in the control of angiogenesis. Both stimulatory and inhibitory effects of perlecan on FGF-2 signaling have been reported. To understand the in vivo function of HS carried by perlecan, the perlecan gene heparan sulfate proteoglycan 2 (Hspg2) was mutated in mouse by gene targeting. The HS at the NH(2) terminus of perlecan was removed while the core protein remained intact. Perlecan HS-deficient (Hspg2(Delta3/Delta3)) mice survived embryonic development and were apparently healthy as adults. However, mutant mice exhibited significantly delayed wound healing, retarded FGF-2-induced tumor growth, and defective angiogenesis. In the mouse corneal angiogenesis model, FGF-2-induced neovascularization was significantly impaired in Hspg2(Delta3/Delta3) mutant mice. Our results suggest that HS in perlecan positively regulates the angiogenesis in vivo.

Animals↗
PubMed2004

Deletion of laminin-8 results in increased tumor neovascularization and metastasis in mice.

Laminin-8 (alpha 4 beta 1 gamma 1) is one of the major laminin isoforms expressed in vascular endothelial basement membranes. Here we show that deletion of laminin-8 in mice affects angiogenesis under pathological conditions. Murine tumor models used in laminin alpha 4-deficient mice results in hyperneovascularization and significant promotion of tumor growth and metastasis. The higher tumor growth rates in mutant mice correlate with decreased tumor cell apoptosis. Depletion of laminin alpha 4 chain may alter the structure of vascular basement membranes, leading to increased angiogenesis. Our data suggest that the laminin-8 plays a critical role in the regulation of pathological angiogenesis.

Animals↗
PubMed2004

An endothelial laminin isoform, laminin 8 (alpha4beta1gamma1), is secreted by blood neutrophils, promotes neutrophil migration and extravasation, and protects neutrophils from apoptosis.

During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.

Aflatoxin B1↗
PubMed2004

Altered NPY and AgRP in membrane type-1 matrix metalloproteinase-deficient mice.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) knockout (KO) mice fail to gain weight and die 3-4 weeks after birth. To understand the wasting phenotype in MT1-MMP-KO mice we studied the expression of some hypothalamic neuropeptides involved in control of appetite and body weight. In MT1-MMP-KO mice, neuronal perikarya in the arcuate nucleus displayed accumulations of NPY and agouti-related protein (AgRP) immunoreactivity (-ir). In contrast, NPY-ir and AgRP-ir were reduced in the projection areas of the arcuate neurons. NPY and AgRP are known to relay metabolic signals from the periphery into the brain to stimulate body weight gain. Their altered subcellular distribution suggests that MT1-MMP is involved in postnatal development of the arcuate NPY/AgRP-system which may contribute to the generation of the wasting phenotype.

Agouti-Related Protein↗
PubMed2004

Tissue expression of nephrin in human and pig.

Nephrin is a major component of the glomerular filtration barrier. Mutations in the nephrin gene (NPHS1) are responsible for congenital nephrotic syndrome of the Finnish type (NPHS1). Nephrin was at first thought to be podocyte specific, but recent studies have suggested that nephrin is also expressed in nonrenal tissues such as pancreas and CNS. We studied the expression of nephrin in human and porcine tissues at different stages of development and correlated these findings to clinical characteristics of NPHS1 children. Immunofluorescence staining and Western blotting were used to detect nephrin protein in frozen tissue samples. Polyclonal antibodies against the intracellular part of nephrin were used in these analyses. In situ hybridization was used to detect nephrin mRNA in specimens from normal human subjects and patients with NPHS1. Nephrin protein was not detected in nonrenal tissues obtained from human and porcine fetuses, newborns, and infants. Likewise, nephrin mRNA expression was not observed outside kidney glomerulus in normal or NPHS1 children. The phenotype analysis of NPHS1 children with severe nephrin gene mutations supported the findings in the tissue expression studies and revealed no impairment of the neurologic, testicular, or pancreatic function in a great majority of the patients. The studies suggest that nephrin has no major clinical significance outside the kidney.

Animals↗
PubMed2004

Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan.

Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.

Animals↗
PubMed2004

Monoclonal antibodies to human nephrin.

Nephrin is a 180-200-kDa transmembrane protein of the immunoglobulin superfamily. In the kidney, nephrin localizes to the slit diaphragm (SD) between interdigitating podocyte foot processes and mutations in the nephrin gene cause congenital nephrotic syndrome. In addition to this rare genetic disorder, recent reports indicate that nephrin is more generally involved in the pathogenesis of glomerular disease. In this report, we describe production and characterization of mouse monoclonal antibodies to human nephrin, and discuss their applications. Recombinant human nephrin variants were produced in both prokaryotic and eukaryotic expression systems and purified proteins were used in an immunization protocol. A total of 16 antibodies were characterized for their reactivity with the nephrin by using ELISA, Western blots, immunoprecipitation and immunostaining of frozen and formaldehyde-fixed paraffin embedded tissue sections. The antibody epitopes were mapped using a variety of recombinant human nephrin variants. The detailed screening and characterization proved to be essential in order to find the most suitable antibody for each application. These antibodies will find wide use in studies of human nephrin and its involvement in kidney disease.

Antibodies, Monoclonal↗
PubMed2004

Defective trafficking of nephrin missense mutants rescued by a chemical chaperone.

The nephrin gene (NPHS1) is mutated in congenital nephrotic syndrome of the Finnish type. Most mutations found in non-Finnish patients are missense mutations. The most common consequence of missense mutations in congenital nephrotic syndrome is a defect in intracellular transport and retention of the mutant proteins in the endoplasmic reticulum (ER), possibly as a result of misfolding and unfavored conformation. Because sodium 4-phenylbutyrate has been shown to function as a chemical chaperone and to correct the cellular trafficking of several mislocalized or misfolded mutant plasma membrane proteins, the effects of this compound on the missense mutants identified in patients with congenital nephrotic syndrome of the Finnish type were investigated. This study was performed using human embryonic kidney 293 cells stably expressing wild-type or missense nephrin mutants trapped in the ER. Immunofluorescence microscopy and cell surface biotinylation showed that treatment with sodium 4-phenylbutyrate rescued several of the missense mutants from the ER to the cell surface. All of the rescued mutants were found to be able to interact with Neph1. Furthermore, their tyrosine phosphorylation was rapidly induced by clustering with anti-nephrin antibodies, suggesting that the rescued mutants may be functionally intact.

Biological Transport↗
PubMed2004

Laminin-5 gamma2 chain expression facilitates detection of invasive squamous cell carcinoma of the uterine cervix.

Because it has been suggested that laminin-5 can be used as a sensitive marker for epithelial cell invasion, specimens from patients with invasive squamous cell carcinoma of the uterine cervix with a previous history of preinvasive lesions were evaluated by a newly developed monoclonal antibody directed against the gamma2 chain of laminin-5. Thirty-two archival paraffin specimens consisting of the matched preinvasive and invasive lesions from 15 women were evaluated to determine whether gamma2 chain laminin-5 staining was present in lesions that progressed to invasive cancer. With the exception of one tumor (a small cell nonkeratinizing squamous carcinoma), all squamous cell carcinomas exhibited positive staining. Five of 17 preinvasive lesions also were immunoreactive for the laminin-5 protein. A blinded histologic reevaluation revealed invasion or lesions suspicious for invasion in four of five preinvasive lesions. Our findings suggest that laminin-5 determined by a monoclonal antibody facilitates the identification of invasive lesions that are difficult or impossible to identify on routinely stained histologic sections.

Antibodies, Monoclonal↗
PubMed2004

Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane.

BACKGROUND: Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin. METHODS: Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK)293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin. RESULTS: Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules. CONCLUSION: In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.

Cell Line↗