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Billy G. Hudson - One of the best experts on this subject based on the ideXlab platform.
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cellular origins of Type IV Collagen networks in developing glomeruli
Journal of The American Society of Nephrology, 2009Co-Authors: Dale R Abrahamson, Billy G. Hudson, Larysa Stroganova, Dorinbogdan Borza, Patricia L St JohnAbstract:Laminin and Type IV Collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different Type IV Collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize Collagen alpha 1 alpha 2 alpha1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain Collagen alpha 3 alpha 4 alpha 5(IV). To evaluate the origins of Collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildType mice. Hybrid glomeruli within grafts containing numerous host-derIVed, wildType endothelial cells never expressed Collagen alpha 3 alpha 4 alpha 5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-Collagen alpha 5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusIVely within podocytes. Together, these results indicate that Collagen alpha 3 alpha 4 alpha 5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell Type.
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alport s syndrome goodpasture s syndrome and Type IV Collagen
The New England Journal of Medicine, 2003Co-Authors: Billy G. Hudson, Karl Tryggvason, Munirathinam Sundaramoorthy, Eric G NeilsonAbstract:Defects in Type IV Collagen, a Collagenous protein involved in the formation of basement membranes, have been implicated in hereditary Alport's syndrome and acquired Goodpasture's syndrome. Mutations in genes corresponding to the building blocks of Type IV Collagen cause Alport's syndrome, whereas autoantibodies against structures that are usually hidden in the recesses of Collagen IV cause Goodpasture's syndrome.
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autoreactIVe t cells in goodpasture s syndrome recognize the n terminal nc1 domain on α3 Type IV Collagen
Kidney International, 1996Co-Authors: Frank Merkel, Billy G. Hudson, Raghuram Kalluri, Eric G Neilson, Martin Marx, Uwe Enders, Stefan Stevanovic, Gerhard Giegerich, Hans Rammensee, Manfred WeberAbstract:AutoreactIVe T-cells in Goodpasture's syndrome recognize the N-terminal NC1 domain on α3 Type IV Collagen. Goodpasture's syndrome is mediated by immunopathogenic autoantibodies to the α3 NC1 domain of Type IV Collagen. It is not known whether collaborating T-cells participate in this autoreactIVe response. Here we describe the first T-cell clone isolated from a Goodpasture patient autoreactIVe to α3 Type IV Collagen of glomerular basement membrane. To investigate cellular autoreactIVity, T-cells from Goodpasture patients or controls were isolated and stimulated by purified natIVe or recombinant Type IV Collagen proteins and synthetic oligopeptides. Cell surface markers, the T-cell receptor repertoire, and MHC-restriction were analyzed. T-cell clones specific for the α3(IV) NC1 domain were established in two Goodpasture patients, but not in controls. One of the three CD8 + T-cell clones was characterized further. It was MHC class I restricted (HLA-A11) and expressed the T-cell receptor Vβ 5.1. chain. This clone specifically recognized a motif at the N-terminal area of the α3(IV) NC1 domain (AA 51 to 59: GSPATWTTR). We conclude that autoreactIVe T-cells exists in Goodpasture patients and may play a crucial role in the inflammatory process. T-cell clones are autoreactIVe to the α3(IV) NC1 domain. At least for one of the clones, the T-cell epitope is different from the putatIVe antibody-binding site.
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the alpha 3 chain of Type IV Collagen induces autoimmune goodpasture syndrome
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Raghuram Kalluri, Vincent H Gattone, Milton E Noelken, Billy G. HudsonAbstract:Abstract Human Goodpasture syndrome is a lethal form of autoimmune disease that is characterized by pulmonary hemorrhage and glomerulonephritis. The tissue injury is mediated by autoantibodies that bind to glomerular and alveolar basement membrane. The target autoantigen is alpha 3(IV) Collagen, one of six genetically distinct chains that comprise Type IV Collagen, and the epitope is sublocalized to the nonCollagenous domain (NC1) of the alpha 3 chain. The present study reports the unique capacity of alpha 3(IV)NC1 dimer from bovine kidney to aberrantly engage the immune system of rabbits to respond to self, mimicking the organ-specific form of the human disease, whereas the other chains of Type IV Collagen are nonpathogenic. However, alpha 3(IV)NC1 hexamer was nonpathogenic, suggesting the exposure of a pathogenic epitope upon dissociation of hexamer into dimers. Exposure of the pathogenic epitope by infection or organic solvents, events which are thought to precede Goodpasture syndrome, may be the principal factor in the etiology of the disease. The pathogenicity of alpha 3(IV) Collagen brings full circle a decade of research that has identified four novel chains (alpha 3-alpha 6) of Type IV Collagen.
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Type IV Collagen of engelbreth holm swarm tumor matrix identification of constituent chains
Connective Tissue Research, 1992Co-Authors: Billie J Wisdom, Sripad Gunwar, Milton E Noelken, Mark D Hudson, Billy G. HudsonAbstract:The nonCollagenous hexamer (NC1) domain of Type IV Collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the fIVe known kinds of alpha(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derIVed exclusIVely from the alpha 1 and alpha 2 chains. Western blots of hexamer subunits confirmed the sequence results, as the subunits. identified as alpha 1(IV) and alpha 2(IV) NC1 domains reacted with antibodies directed specifically against those subunits. Conversely, no reactIVity of NC1 hexamer subunits was seen with Goodpasture serum, or with antibodies directed specifically against the alpha 3, alpha 4, and alpha 5 NC1 domains, confirming the lack of alpha 3, alpha 4, and alpha 5 chains. These results revealed that the Type IV Collagen component of the EHS sarcoma matrix is comprised exclusIVely of alpha 1 and alpha 2 chains. Its relatIVe homogeneity simplifies, but restricts, interpretation of studies that employ it as a model Type IV Collagen because the studies would be based only on alpha 1 and alpha 2 chains.
Hans Peter Bachinger - One of the best experts on this subject based on the ideXlab platform.
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post translational modification of Type IV Collagen with 3 hydroxyproline affects its interactions with glycoprotein vi and nidogens 1 and 2
Journal of Biological Chemistry, 2018Co-Authors: Nathan T Montgomery, Keith Zientek, Elena Pokidysheva, Hans Peter BachingerAbstract:: Type IV Collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV Collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp's biochemical role in Type IV Collagen's interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of Collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between Type IV Collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to Type IV Collagen without 3-Hyp is novel. Using tandem MS, we show that the putatIVe glycoprotein VI-binding site is 3-Hyp-modified in WT PFHR-9 Type IV Collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of Type IV Collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp-modifying enzyme of Type IV Collagen. These findings underscore the importance of post-translational modifications of Type IV Collagen for interactions with other proteins.
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biological role of prolyl 3 hydroxylation in Type IV Collagen
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Elena Pokidysheva, Keith Zientek, Sergei P Boudko, Janice A Vranka, Kerry Maddox, Markus Moser, Reinhard Fassler, Jerry Ware, Hans Peter BachingerAbstract:Collagens constitute nearly 30% of all proteins in our body. Type IV Collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper Collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non–3-hydroxylated embryonic Type IV Collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenoType is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial Collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In Type IV Collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of Type IV Collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non–3-hydroxylated Type IV Collagen might also play a role in the progression of malignant tumors and metastasis.
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biological role of prolyl 3 hydroxylation in Type IV Collagen
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Elena Pokidysheva, Keith Zientek, Sergei P Boudko, Janice A Vranka, Kerry Maddox, Markus Moser, Reinhard Fassler, Jerry Ware, Hans Peter BachingerAbstract:Collagens constitute nearly 30% of all proteins in our body. Type IV Collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper Collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non–3-hydroxylated embryonic Type IV Collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenoType is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial Collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In Type IV Collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of Type IV Collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non–3-hydroxylated Type IV Collagen might also play a role in the progression of malignant tumors and metastasis.
Raghu Kalluri - One of the best experts on this subject based on the ideXlab platform.
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chapter 1 molecular mechanism of Type IV Collagen derIVed endogenous inhibitors of angiogenesis
Methods in Enzymology, 2008Co-Authors: Vesselina G Cooke, Raghu KalluriAbstract:Angiogenesis, the process of new blood vessel formation, is regulated on both genetic and molecular levels. Pro- and anti-angiogenic stimuli maintain the angiogenic balance, and the tipping of that balance toward pro-angiogenic actIVity is critical for tumor growth and survIVal. Endogenous inhibitors of angiogenesis, many of which are fragments from large extracellular matrix proteins, counter the effect of growth factors and keep angiogenesis in check. This chapter will discuss the molecular mechanisms of endogenous inhibitors derIVed from Type IV Collagen and review the in vitro and in vIVo assays available to study their role in angiogenesis.
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Type IV Collagen α6 chain derIVed nonCollagenous domain 1 α6 IV nc1 inhibits angiogenesis and tumor growth
International Journal of Cancer, 2007Co-Authors: Thomas M Mundel, Hikaru Sugimoto, Anna Maija Yliniemi, Yohei Maeshima, Mark W Kieran, Raghu KalluriAbstract:Type IV Collagen is a major component of vascular basement membranes. The nonCollagenous (NC1) domains of several α-chains of Type IV Collagen reveal a capacity to inhibit angiogenesis and tumor growth. Here, we demonstrate that the NC1 domain of the α6 chain of Type IV Collagen (α6NC1) is an endogenous inhibitor of angiogenesis and tumor growth. Recombinant α6NC1 inhibits human endothelial cell proliferation and neovascularization of Matrigel plugs in mice. The α6NC1 suppresses the growth of subcutaneously transplanted Lewis lung carcinoma and also spontaneous pancreatic insulomas that develop in the Rip1Tag2 mice. Inhibition of tumor growth is associated with significantly diminished microvascular density. CollectIVely, our results demonstrate that α6NC1 is an endogenous inhibitor of angiogenesis and tumor growth. © 2007 Wiley-Liss, Inc.
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Type IV Collagen derIVed angiogenesis inhibitors
Microvascular Research, 2007Co-Authors: Thomas M Mundel, Raghu KalluriAbstract:The concept of anti-angiogenesis therapy was introduced by Judah Folkman in 1971 and since then, a plethora of pro- and anti-angiogenic factors have been identified. In the recent years, it has become clear that angiogenesis, the formation of new capillaries from a pre-existing capillary network, is highly regulated by the action of pro- and anti-angiogenic factors. In the healthy adult organism the "angiogenic-switch" is likely turned "Off", i. e. anti-angiogenic factors are likely counteracting the pro-angiogenic factors resulting in a non-angiogenic state. Angiogenesis is encountered during wound healing processes, the female menstrual cycle and endometrial remodeling, as well as during embryonic development and organ growth. In the pathological setting, angiogenesis plays an important role in different diseases like rheumatoid arthritis, psoriasis, macular degeneration, diabetic retinopathy, and tumor growth. In this regard, recent studies have described several endogenous inhibitors of angiogenesis, with a subset derIVed from extracellular matrix (ECM) proteins. This review will particularly focus on the Type IV Collagen-derIVed angiogenesis inhibitors Arresten, Canstatin and Tumstatin.
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human α1 Type IV Collagen nc1 domain exhibits distinct antiangiogenic actIVity mediated by α1β1 integrin
Journal of Clinical Investigation, 2005Co-Authors: Hikaru Sugimoto, Dominic Cosgrove, Akulapalli Sudhakar, Pia Nyberg, Venkateshwar G Keshamouni, Arjuna P Mannam, Raghu KalluriAbstract:Human nonCollagenous domain 1 of the α1 chain of Type IV Collagen [α1(IV)NC1], or arresten, is derIVed from the carboxy terminal of Type IV Collagen. It was shown to inhibit angiogenesis and tumor growth in vIVo; however, the mechanisms involved are not known. In the present study we demonstrate that human α1(IV)NC1 binds to α1β1 integrin, competes with Type IV Collagen binding to α1β1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, α1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK actIVation in ECs but had no effect on the PI3K/Akt pathway. In contrast, α1(IV)NC1 did not affect proliferation, migration, or the actIVation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in α1 integrin receptor knockout ECs. Consistent with this, α1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vIVo but failed to do the same in α1 integrin receptor knockout mice. This suggests a highly specific, α1β1 integrin–dependent antiangiogenic actIVity of α1(IV)NC1. In addition, α1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1α and VEGF in ECs cultured on Type IV Collagen by inhibiting ERK1/2 and p38 actIVation. This unravels a hitherto unknown function of human α1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. CollectIVely, the above data indicate that α1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.
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distinct antitumor properties of a Type IV Collagen domain derIVed from basement membrane
Journal of Biological Chemistry, 2000Co-Authors: Yohei Maeshima, Pablo C Colorado, Adriana Torre, Kathryn A Holthaus, James A Grunkemeyer, Mark B Ericksen, Helmut Hopfer, Yingwen Xiao, Isaac E Stillman, Raghu KalluriAbstract:Abstract Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV Collagen is a major component of vascular basement membrane, and recently we identified a fragment of Type IV Collagen α2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209–1215). In the present study we characterize two different antitumor actIVities associated with the nonCollagenous 1 (NC1) domain of the α3 chain of Type IV Collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185–203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395–20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vIVo assays. The α3(IV)NC1 inhibited in vIVoneovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vIVo endothelial cell-specific apoptosis. The anti-angiogenic actIVity was localized to amino acids 54–132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185–203 amino acid region responsible for the antitumor cell actIVity. Additionally, our experiments indicate that the antitumor cell actIVity is not realized until the peptide region is exposed by truncation of the α3(IV)NC1 domain, a requirement not essential for the anti-angiogenic actIVity of this domain. CollectIVely, these results effectIVely highlight the distinct and unique antitumor properties of the α3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.
Clifford E Kashtan - One of the best experts on this subject based on the ideXlab platform.
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familial hematuria due to Type IV Collagen mutations alport syndrome and thin basement membrane nephropathy
Current Opinion in Pediatrics, 2004Co-Authors: Clifford E KashtanAbstract:Recent molecular genetic studies have shown that mutations in Type IV Collagen account for a significant proportion of patients with persistent glomerular hematuria. This review will discuss the implications of these findings for the diagnosis and management of persistent glomerular hematuria. Type IV Collagen mutations are associated with a continuum of disease severity. Heterozygous mutations typically cause isolated, nonprogressIVe hematuria. Mutations in both alleles of the autosomal Type IV Collagen genes, or hemizygous mutations in the X-linked gene encoding the α5 chain of Type IV Collagen, result in progressIVe renal disease that is often associated with sensorineural deafness (Alport syndrome). Animal models of Alport syndrome have begun to provide insights into the pathogenesis of end-stage renal disease in Alport syndrome, with potentially important implications for therapy. Recognition that glomerular hematuria frequently has a genetic basis is important for accurate genetic counseling, early identification of indIViduals at risk for end-stage renal disease development, and institution of therapies to delay the onset of ESRD.
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familial hematuria due to Type IV Collagen mutations alport syndrome and thin basement membrane nephropathy
Current Opinion in Pediatrics, 2004Co-Authors: Clifford E KashtanAbstract:Purpose of review Recent molecular genetic studies have shown that mutations in Type IV Collagen account for a significant proportion of patients with persistent glomerular hematuria. This review will discuss the implications of these findings for the diagnosis and management of persistent glomerular hematuria. Recent findings Type IV Collagen mutations are associated with a continuum of disease severity. Heterozygous mutations typically cause isolated, nonprogressIVe hematuria. Mutations in both alleles of the autosomal Type IV Collagen genes, or hemizygous mutations in the X-linked gene encoding the alpha 5 chain of Type IV Collagen, result in progressIVe renal disease that is often associated with sensorineural deafness (Alport syndrome). Animal models of Alport syndrome have begun to provide insights into the pathogenesis of end-stage renal disease in Alport syndrome, with potentially important implications for therapy. Summary Recognition that glomerular hematuria frequently has a genetic basis is important for accurate genetic counseling, early identification of indIViduals at risk for end-stage renal disease development, and institution of therapies to delay the onset of ESRD.
Alessandra Balduini - One of the best experts on this subject based on the ideXlab platform.
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megakaryocytes contribute to the bone marrow matrix environment by expressing fibronectin Type IV Collagen and laminin
Stem Cells, 2014Co-Authors: Alessandro Malara, Manuela Currao, Cristian Gruppi, Giuseppe Celesti, Gianluca Viarengo, Chiara Buracchi, Luigi Laghi, David L Kaplan, Alessandra BalduiniAbstract:Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, Type IV Collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vIVo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with Type IV Collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derIVed from fetal lIVer progenitor cells. In vIVo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only Type IV Collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration. Stem Cells 2014;32:926–937
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megakaryocytes contribute to the bone marrow matrix environment by expressing fibronectin Type IV Collagen and laminin
Stem Cells, 2014Co-Authors: Alessandro Malara, Manuela Currao, Cristian Gruppi, Giuseppe Celesti, Gianluca Viarengo, Chiara Buracchi, Luigi Laghi, David L Kaplan, Alessandra BalduiniAbstract:Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, Type IV Collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vIVo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with Type IV Collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derIVed from fetal lIVer progenitor cells. In vIVo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only Type IV Collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration.
