Extracellular Matrix Protein Matrilin-4 Regulates HSC Stress Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 601-601
Author(s):  
Hannah Uckelmann ◽  
Sandra Blaszkiewicz ◽  
Marieke Essers
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Bone Marrow Cells ◽  
Blood System ◽  
Extracellular Matrix Protein

Abstract The life-long maintenance of the blood system is accomplished by a pool of self-renewing multipotent hematopoietic stem cells (HSCs). Adult HSCs are found in a dormant state for most of their lifetime, entering cell cycle only to maintain homeostatic blood supply. Under stress conditions such as infection or chemotherapy, the loss of mature blood cells leads to an activation of dormant HSCs to replenish the blood system. Gene expression analysis performed by our group now revealed that Matrilin-4 is highly expressed in long-term HSCs (LT-HSCs) compared to short-term HSCs or committed progenitors, suggesting a potential role of Matrilin-4 in HSC function. Matrilin-4 is a member of the von Willebrand factor A-containing family of extracellular adapter proteins, which form filamentous structures outside of cells. Using mice lacking the entire family of Matrilins (1-4) we have investigated the role of Matrilins in HSC function. Constitutive Matrilin 1-4 KO mice exhibit normal hematopoiesis with a mild reduction in bone marrow cellularity and LSK numbers. However, when Matrilin KO bone marrow cells are pushed to proliferate in competitive transplantation assays with wildtype (WT) cells, they show a striking growth advantage. In a competitive transplant setting, where bone marrow cells of Matrilin KO versus WT mice are transplanted in a 1:1 ratio, the KO cells outcompete WT cells within four weeks, reaching a 90% chimerism at 16 weeks. This competitive advantage of Matrilin KO cells is evident in the long-term stem cell level as well as progenitors and is consistent in secondary transplants. To explore this remarkable phenotype, we performed single cell transplantation experiments of LT-HSCs and observed a more rapid reconstitution of peripheral blood cell levels of KO HSCs compared to WT controls. Confirming this growth advantage, Matrilin KO LSK cells show higher colony forming and serial replating potential in vitro, which can be rescued by the addition of recombinant or overexpressed Matrilin-4. While Matrilin-4 is highly expressed in homeostatic HSCs, in vivo treatment with IFNα or other inflammatory agents, such as LPS or G-CSF result in a dramatic downregulation (25-fold) of Matrilin-4 on the transcript as well as the protein level. Moreover, Matrilin KO HSCs are more sensitive to inflammatory stress, as they show a 2-fold stronger cell cycle activation in response to IFNα in vivo. Critically, Matrilin-4 KO HSCs return to the G0 state of the cell cycle normally after stress-induced activation and transplantation, thereby preventing their exhaustion. In summary, we show that the extracellular matrix protein Matrilin-4 is a novel component of the HSC niche, regulating HSC stress response. Surprisingly, HSCs lacking this extracellular matrix protein show a higher HSC potential due to an accelerated response to stress. Our data suggest that high expression of Matrilin-4 in LT-HSCs confers a resistance to stress stimuli. In situations of acute stress such as infection or transplantation however, this protection is rapidly lost to allow HSCs to efficiently replenish the blood system. Disclosures No relevant conflicts of interest to declare.

P6296The role of extracellular matrix protein 1 (ECM1) - a novel link between inflammation and cardiac fibrosis

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Hardy ◽  
N S Mabotuwana ◽  
L A Murtha ◽  
B Coulter ◽  
S S Bezenilla ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Cardiac Fibrosis ◽  
In Situ Hybridisation ◽  
Extracellular Matrix Protein ◽  
Cardiac Diseases ◽  
Extracellular Matrix Protein 1 ◽  
Primary Mouse

Abstract Introduction Cardiac fibrosis is a severe consequence of cardiovascular disease and aging, in which we currently have no effective treatments. The mechanisms underpinning the development of cardiac fibrosis remains poorly understood. Our preliminary data suggested extracellular matrix protein 1 (ECM1) is involved in cardiac fibrosis. We therefore aimed to investigate the role of ECM1 in several fibrotic cardiac diseases. Methods Young and ageing (3m/18m) male C57BL/6 mice, and primary mouse cardiac fibroblast (cFB) cultures, commercial human cardiac fibroblasts (Hu-cFB), human coronary artery endothelial cell (HCAEC)/smooth muscle cell (HCASMC), and human cardiac myocyte (HCM) cell lines were used. Young mice were subject to myocardial infarction (MI, 3-day/28-day, n=6/6), or pressure overload (TAC, 3-day/13-week, n=4/4). Left ventricle (LV) was collected at all time-points, and at 18m (ageing; n=3). Spleen and bone marrow was extracted from young control mice. Hu-cFB cells were treated with recombinant ECM1 (20ng/ml) for either 10, 30 or 50 min, or 48h. Immunoblotting was conducted on all samples, qPCR on LV tissue only, density gradient centrifugation and multicolour flow cytometry coupled with fluorescent ECM1 mRNA in-situ hybridisation (FISH-Flow) on bone marrow cells. Results ECM1 expression was upregulated in ageing LV (mRNA 2.2±0.1-fold, p=0.0002; protein 2.0-fold, p=0.0006), day-3 post-MI (mRNA, 4.9±2.0-fold, p=0.004; protein, 3.0-fold, p=0.004), a trend of ECM1 upregulation was observed at day-28 post-MI (mRNA, 13.2±12.0-fold, p=0.003; protein, 1.8-fold, p=0.2), but no change post-TAC. Both ERK1/2 and AKT phosphorylation was upregulated 10 min post-ECM1 treatment of Hu-cFBs (ERK1/2, 2.0-fold, p<0.0001; AKT, 1.9-fold, p<0.0001), and Collagen-I protein expression was upregulated 48h post-ECM1 treatment (1.9-fold, p=0.004). ECM1 protein was not expressed in cFB, Hu-cFB, HCAEC, HCASMC or HCM, however ECM1 protein was highly expressed in spleen and bone marrow; to a greater extent in granulocytes compared to monocytes (p=0.004). tSNE analysis of ECM1 mRNA FISH-Flow revealed ECM1+ are highly granular, moderate to large in size, and express (to varying levels) CD45, CD11b, CD11c, F4/80, Ly6-C, Ly-6G, and FcεrI-α. However ECM1+ cells did not express markers indicative of smaller cells (CD3 or MHC II). Conclusions These data demonstrate that ECM1 plays a role in ageing and post-MI fibrosis. Although ECM1 was not produced by resident cardiac cells, it was highly expressed in spleen and bone marrow; specifically, large, granular bone marrow cell sub-types such as granulocytes and/or macrophages. Our data suggest ECM1 is expressed by cardiac infiltrating leukocytes to provoke fibroblast collagen expression in a disease specific manner; potentially via the ERK1/2 and/or AKT pathway activation. Therefore, ECM1 warrants further investigation, and may be a promising target for the treatment of fibrotic cardiac diseases. Acknowledgement/Funding John hunter hospital charitable trust, Hunter medical research institute (HMRI) grants

scholarly journals Inflammation Mediated Bone Marrow Remodeling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-2-SCI-2
Author(s):  
Marieke A. G. Essers
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Matrix Protein ◽  
The Body ◽  
Extracellular Matrix Protein ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Inflammatory Stress

Infection is a common, natural form of stress, with which the body is regularly challenged. During infection or inflammation, cells of the immune system are responsible for fighting the invading pathogens. This leads to consumption of blood and immune cells due to mobilization of these cells to the site of infection, or by apoptosis as part of the host response to invading pathogens. Restoration of the balance of the hematopoietic system following successful elimination of the infection is a crucial part of the recovery of the body. In addition, both clinical and experimental data indicate that depending on the scale and duration, infection and inflammation can induce hematopoietic dysfunction compromising immune defense mechanisms and possibly contributing to the development of hematologic malignancies. Restoring the balance of the hematopoietic system depends on the replacement of lost immune cells by the activity of hematopoietic stem cells (HSCs). During the last ten years we and others could show that this so-called emergency hematopoiesis is driven by pro-inflammatory cytokines, who are increasingly produced upon infection or inflammation in the bone marrow and can directly drive the activation of normally quiescent HSCs. An interesting observation from these data is the often opposing impact of these pro-inflammatory cytokines on HSCs in vivo versus in vitro. Whereas in vivo treatment of mice with for example IFNα leads to a strong increase in proliferation of the HSCs, in vitro treatment with IFNα inhibits the HSCs. Furthermore, data from viral infection experiments have shown sustained alterations in the inflammatory cytokine/chemokine profile in the bone marrow weeks after the infection. All these data suggest that interactions of HSCs with their direct environment or signals from this environment are important for a proper response of the HSCs during environmental stress. Research in recent years has focused on unraveling the different components of the HSC stem cell niche. However, the molecular and cellular basis of the BM HSC niche, and signals exchanged between HSC and niche cells under stress conditions remain poorly understood. We initially focused on how the niche responds to inflammatory stress, and could show that the BM stem cell vascular niche was remodeled in response to IFNα. IFNα treatment of mice resulted in increased BM vascularity, expression of key inflammatory and endothelial-stimulatory markers on ECs and increased BM vascular leakiness. These data indicate a novel acute response of the BM vasculature to primary inflammatory signaling, suggesting alterations of the HSC niche in response to stress. ECs are not the only cells in the BM niche responding to inflammatory stress. Using different mouse models and single cell sequencing technology we are currently not only investigating the impact of inflammatory stress on the other components of the niche but also try to unravel the possible changes in interactions and signals between the HSCs and the niche. One example is our data on the role of the extracellular matrix protein Matrilin-4 (Matn4) in the regulation of the HSC response. Under homeostasis high expression of Matn-4 in HSCs confers a resistance to stress stimuli. In situations of acute stress, such as an infection or transplantation, this protection is rapidly lost through down-regulation of Matn-4, allowing HSCs to efficiently replenish the blood system. Thus, these data indicate an important role for the control of the interactions of HSCs with the extracellular matrix in regulating the HSC stress response in vivo. In summary, investigating the response of the bone marrow niche and the role of stem cell-ECM-niche interactions in controlling the HSC stress response will help us to better understand the shortterm and longterm impact of infection and inflammation on the HSCs and their niche. Potential Articles of Interest: Hirche C, Frenz T, Haas S, et al (2017). Systemic Virus Infections differentially modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo. Cell Reports19: 2345-56Velten L, Haas SF, Raffel S, et al (2017). Human haematopoietic stem cell lineage commitment is a continuous process. Cell Biol.19: 271-281Prendergast AM, Kuck A, van Essen M, et al (2017). IFNa mediated remodeling of endothelial cells in the bone marrow niche. Haematologica,102: 445-453Uckelmann H, Blaszkiewicz S, Nicolae C, et al (2016). Extracellular matrix protein Matrilin-4 regulates stress-induced HSC proliferation via CXCR4. J Exp. Med.213: 1961-1971Haas S, Hansson J, Klimmeck D, et al (2015). Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors. Cell Stem Cell17: 422-34 Disclosures No relevant conflicts of interest to declare.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system

1996 ◽  
Vol 109 (7) ◽  
pp. 1749-1757 ◽  
Author(s):  
N. Soussi-Yanicostas ◽  
J.P. Hardelin ◽  
M.M. Arroyo-Jimenez ◽  
O. Ardouin ◽  
R. Legouis ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Extracellular Matrix ◽  
Nervous System ◽  
Cell Membrane ◽  
Heparan Sulfate ◽  
Matrix Protein ◽  
Neuronal Cell ◽  
Extracellular Matrix Protein ◽  
Cell Populations

The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.

scholarly journals LTBP1 promotes fibrillin incorporation into the extracellular matrix

2021 ◽  
Author(s):  
Matthias Przyklenk ◽  
Veronika Georgieva ◽  
Fabian Metzen ◽  
Sebastian Mostert ◽  
Birgit Kobbe ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Wide Spectrum ◽  
Extracellular Matrix Protein ◽  
Connective Tissues ◽  
Pathogenic Variants ◽  
Human Pathology ◽  
Fibrillin 1

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFβ growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFβ-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

scholarly journals Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1460-1462 ◽  
Author(s):  
ME Pietrzyk ◽  
GV Priestley ◽  
NS Wolf
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Improved Method ◽  
Hematopoietic Stem ◽  
Infusion Study ◽  
A Cell ◽  
Cell Subpopulations ◽  
Over Time

It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14- day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

scholarly journals Silencing long non-coding RNA XIST suppresses drug resistance in acute myeloid leukemia through down-regulation of MYC by elevating microRNA-29a expression

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Chong Wang ◽  
Lingling Li ◽  
Mengya Li ◽  
Weiqiong Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Cellular Viability ◽  
Down Regulation ◽  
Acute Myeloid

Abstract Background Long non-coding RNAs (lncRNAs) are biomarkers participating in multiple disease development including acute myeloid leukemia (AML). Here, we investigated molecular mechanism of X Inactive-Specific Transcript (XIST) in regulating cellular viability, apoptosis and drug resistance in AML. Methods XIST, miR-29a and myelocytomatosis oncogene (MYC) expression in AML bone marrow cells collected from 62 patients was evaluated by RT-qPCR and Western blot analysis. Besides, the relationship among XIST, miR-29a and MYC was analyzed by dual luciferase reporter assay, RIP, and RNA pull down assays. AML KG-1 cells were treated with anti-tumor drug Adriamycin. The role of XIST/miR-29a/MYC in cellular viability, apoptosis and drug resistance in AML was accessed via gain- and loss-of-function approaches. At last, we evaluated role of XIST/miR-29a/MYC on tumorigenesis in vivo. Results XIST and MYC were up-regulated, and miR-29a was down-regulated in AML bone marrow cells. Silencing XIST inhibited cellular activity and drug resistance but promoted cellular apoptosis of KG-1 cells by down-regulating MYC. XIST inhibited miR-29a expression to up-regulate MYC. Moreover, silencing XIST inhibited tumorigenesis of AML cells in vivo. Conclusions Overall, down-regulation of XIST decreased MYC expression through releasing the inhibition on miR-29a, thereby reducing drug resistance, inhibiting viability and promoting apoptosis of AML cells.

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 829-836 ◽  
Author(s):  
Timothy W. Austin ◽  
Suzan Salimi ◽  
Gabor Veres ◽  
Franck Morel ◽  
Heini Ilves ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

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