Activation of Epac stimulates integrin-dependent homing of progenitor cells

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2640-2646 ◽  
Author(s):  
Guillaume Carmona ◽  
Emmanouil Chavakis ◽  
Ulrike Koehl ◽  
Andreas M. Zeiher ◽  
Stefanie Dimmeler
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Matrix Protein ◽  
Therapeutic Potential ◽  
Limb Ischemia ◽  
Β1 Integrin ◽  
Nucleotide Exchange ◽  
Β2 Integrin ◽  
The Matrix

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However, the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1, a nucleotide exchange protein for Rap1, which is directly activated by cAMP, can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs, CD34+ hematopoietic progenitor cells, and mesenchymal stem cells (MSCs). Specifically, short-term stimulation with a specific Epac activator increased the β2-integrin–dependent adhesion of EPCs to endothelial cell monolayers, and of EPC and CD34+ cells to ICAM-1. Furthermore, the Epac activator enhanced the β1-integrin–dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition, Epac1 activation induced the β1- and β2-integrin–dependent migration of EPCs on fibronectin and fibrinogen. Interestingly, activation of Epac rapidly increased lateral mobility of β1- and β2-integrins, thereby inducing integrin polarization, and stimulated β1-integrin affinity, whereas the β2-integrin affinity was not increased. Furthermore, prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing.

Dendritic Cells Direct Specific Stimulation of Hematopoietic Stem/Progenitor Cells Generating a Potentially Therapeutic Cell Population

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 483-483
Author(s):  
Yael Porat ◽  
Efrat Assa-Kunik ◽  
Michael Belkin ◽  
Shlomo Bulvik
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hind Limb ◽  
Progenitor Cell ◽  
Limb Ischemia ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Medium Group

Abstract Abstract 483 Background: Recent data show that dendritic cells (DCs) are important component of stem cell niches in the bone marrow and spleen, and as such may have a role in stem/progenitor cell homeostasis and in pro- and anti-angiogenic processes (Gabrilovich, 1996; Dikov, 2005; Sozzani, 2007). For the first time we report a process in which human Hematopoietic Stem/Progenitor Cells (HSPC) are specifically stimulated by activated DCs. This newly developed process makes it possible to use even unmobilized blood cells as a source for sufficient numbers of potentially therapeutic stem/progenitor cells, thus eliminating the need for surgical bone marrow harvesting and G-CSF mobilization. Goal: To show that DCs can direct the generation of an Enriched Endothelial Progenitor Cell (EnEPC) population, which includes Endothelial Progenitor Cells (EPC) and HSPCs, addressed to treat blood vessel malfunction. Methods: Samples of 250 ml blood from both healthy and diabetic patients were collected under hospital's IRB (Bulvik 15/150109) and used as the cell source. Selected immature plasmacytoid and myeloid DCs were alternatively activated for 2–24 hours in order to induce pro-angiogenic signals before being co-cultured with HSPCs. Cultures of up to 66 hours resulted in the generation of EnEPC in a formulation named BC1. BC1 was tested in-vitro by FACS, tube formation, colony forming units (CFU) and cytokine secretion tests. In-vivo BC1 was tested in the hind limb ischemia model (Goto, 2006; Kang, 2009) of critical limb ischemia (CLI) in order to evaluate its therapeutic potential, dosing levels and bio-distribution following intramuscular transplantation (IM). The study applied a genetically modified SCID/Nude mice model supporting evaluation of both safety and efficacy of BC1 treatment. A 21-day controlled blinded experiment included a control medium group (N=10); unprocessed cells (PreBC1, N=5); two BC1 groups of 2.5×10^6/mouse, BC1-1 (N=10) cultured for 1day and BC1-3 (N=10) for 3 and a lower cell dose group of 0.5×10^6/Mouse BC1-31 (N=5). Results: DC directed BC1 containing 70 ±5×10^6 cells with a viability of 96.9±1.9% is composed of a mixture of 40.2±11.9% EPC (expressing Ulex-lectin and uptake of AcLDL, CD202b (Tie2), CD309 (VEGGFR-2; KDR), CD31 and VEGFR1) and 29.8±14.3% HSPC (co-expressing CD34 and the migration/homing marker CD184 /CXCR4-R). In-vitro functional tests demonstrated angiogenic and hematopoietic potential and secretion of IL-8, VEGF, and IL-10 but not TNF and IFN. In-vivo BC1 was found efficient and safe in the hind-limb ischemia model. Evaluation of clinical signs revealed an improvement in limb function and score in all BC1 treated groups over the control medium group. BC1 treatment doubled the blood flow (BF) to the legs from an average of 23±5% after injury to an average of 51±3.1% on day 21 after treatment (p<0.005). Conclusions: The presented data show that activated DCs can direct in-vitro cellular interactions resulting in a potentially therapeutic EnEPC population after a short-term culture of HSPC. This process makes it possible to use unmobilized blood as the raw material for generating stem/progenitor cell products. The method described here is far safer for patients and much more convenient for clinicians compared to existing methods, such as G-CSF mobilization or bone marrow and fat cells harvesting. Further research needs to be done in order to test the safety and efficacy of these cells in patients suffering from cardiovascular diseases and blood vessel malfunctions. Disclosures: Porat: BioGenCell: Employment, Equity Ownership, Research Funding; Laniado Hospital: Consultancy. Assa-Kunik:BioGenCell: Employment; Laniado Hospital: Employment. Belkin:BioGenCell: Consultancy, Equity Ownership.

Abstract 1130: Role Of PI3Kγ For Integrin-dependent Adhesive And Migratory Functions Of Progenitor Cells In Vitro And In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Emmanouil Chavakis ◽  
Guillaume Carmona ◽  
Triantafyllos Chavakis ◽  
Andreas M Zeiher ◽  
Stefanie Dimmeler
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Molecular Mechanisms ◽  
Catalytic Subunit ◽  
Β1 Integrin ◽  
Cell Homing ◽  
Integrin Ligand

Endothelial progenitor cells (EPC) are recruited to ischemic regions to improve neovascularization. β1- and β2-integrins play a crucial role for progenitor cell homing to ischemic tissues. Chemokines and their respective G-protein coupled receptors (GPCRs) are involved in the EPC homing to ischemic tissues. The phosphatidylinositol-3-kinase catalytic subunit gamma (PI3Kγ) is the PI3K isoform, which selectively transduces signals from GPCRs. Here, we investigated the role of PI3Kγ for integrin-dependent homing functions of progenitor cells. As assessed by western blot, EPC express the catalytic subunit PI3Kγ. We then studied the role of PI3Kγ for EPC migration. AS-605240 (100 nM), a selective PI3Kγ-inhibitor (Camps M, Nat. Med., 2005), significantly reduced the SDF1- and the IL-8-induced migration and the SDF1-induced transendothelial migration of human EPC. Adhesion is a further essential step during EPC homing to ischemic tissues. In this regard, the PI3Kγ-inhibitor significantly reduced the SDF1-induced adhesion of EPC on HUVEC monolayers by 69 ± 8 % and on ICAM-1, a β2-integrin ligand. However, the PI3Kγ-inhibitor did not affect the SDF1-induced adhesion of EPC on fibronectin, a β1-integrin ligand, suggesting that PI3Kγ in EPC is involved in the regulation of β2-, but not of β 1-integrin-dependent adhesion. In line with these results, inhibition PI3Kγ blocked the SDF1-induced increase of β2-, but not of β1-integrin-affinity in EPC. Beside EPC, the SDF1-induced migration and adhesion on ICAM-1 of murine bone marrow (BM)-derived Lin − progenitor cells from PI3Kγ-deficient mice (PI3Kγ − / − ) were reduced in comparison to wild type (WT) cells. In addition, PI3Kγ-deficiency led to a significant reduction of homing of murine BM-Lin − progenitor cells to ischemic muscles after intravenous infusion in the model of hind limb ischemia in comparison to WT cells (48 ± 8 % inhibition). In conclusion, these data demonstrate that PI3Kγ plays an essential role for the integrin-dependent homing of progenitor cells in vitro and in vivo. The understanding of the molecular mechanisms of progenitor cell homing is essential for the development of new therapeutic strategies in order to improve the efficacy of cell-based therapies in patients with ischemic disorders.

Abstract 77: Fibronectin Signals Through Pim-1 and β1 Integrin in Cardiac Progenitor Cells to Induce Proliferation and Protection

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Mathias H Konstandin ◽  
Haruhiro Toko ◽  
Mirko Volkers ◽  
Mercedes Quintana ◽  
Natalie Gude ◽  
...  
Keyword(s):  
Cell Death ◽  
Cardiac Function ◽  
Progenitor Cells ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Cardiac Progenitor Cells ◽  
Β1 Integrin ◽  
Dependent Manner ◽  
Integrin Receptor

Background: Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Fibronectin (FN), an extracellular matrix protein, is highly expressed during cardiac development. In adult heart FN is a component of the cardiac stem cell niche and re-appears after myocardial infarction (MI). The role of FN signaling in CPC function and cardiac remodeling following MI has not been elucidated. We demonstrate here proliferative and protective effects of FN signaling in CPC as mediated by β1 integrin receptor and cardioprotective serine/threonine kinase Pim1. Methods: Cell death and proliferation of CPCs was measured using propidium iodide and CyQuant assays. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA depletion of targets. FN was localized in heart sections by immunohistochemistry and cardiac function assessed by echocardiography in control and conditional FN knockout (KO) mouse hearts following MI. Results: FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim1 or deletion of β1 integrin receptor. In vivo CPC expansion correlates with FN expression following MI, and CPC localize to regions of up-regulated FN protein in the infarct. Cardiac function in control and KO mice remains equivalent up to 2 weeks post MI, however by 4 weeks KO heart function worsens compared to control as evidenced by ejection fraction values measuring 16.4 +/- 1.5% vs 26.6 +/- 1.8% at 12 weeks post MI in KO and control hearts, respectively. Conclusion: FN provides pro-survival and pro-proliferative effects to CPCs in a Pim1 kinase and β1 integrin dependent manner. CPCs and FN colocalize in vivo in an infarction injury model, while conditional KO of FN in mice leads to further impairment of cardiac function after MI. Taken together these results indicate previously unidentified cardioprotective and regenerative roles for FN in pathologically challenged heart.

scholarly journals Epac1 activation and progenitor-cell homing

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2501-2501
Author(s):  
Domenico Ribatti
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Therapeutic Potential ◽  
Cell Homing

The article by Carmona and colleagues in this issue of Blood demonstrates that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

scholarly journals Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

Blood ◽  
2010 ◽  
Vol 116 (17) ◽  
pp. 3197-3207 ◽  
Author(s):  
Kirsteen J. Campbell ◽  
Mary L. Bath ◽  
Marian L. Turner ◽  
Cassandra J. Vandenberg ◽  
Philippe Bouillet ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Cytotoxic Agents ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Fetal Liver Cells ◽  
Follicular Lymphomas ◽  
The Impact

Abstract Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

scholarly journals Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

2009 ◽  
Vol 83 (19) ◽  
pp. 9875-9889 ◽  
Author(s):  
Elodie Beaumont ◽  
Daniela Vendrame ◽  
Bernard Verrier ◽  
Emmanuelle Roch ◽  
François Biron ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Matrix Protein ◽  
Immunodeficiency Virus ◽  
The Matrix ◽  
Hiv 1

ABSTRACT Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.

Abstract 119: Potential Role for Bone Marrow Niche and Endothelial Progenitor Cell Dysfunction in Critical Limb Ischemia

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Martin Teraa ◽  
Ralf W Sprengers ◽  
Frans L Moll ◽  
Marianne C Verhaar ◽  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Critical Limb Ischemia ◽  
Progenitor Cell ◽  
Limb Ischemia ◽  
Endothelial Damage ◽  
Inverse Association ◽  
Healthy Controls ◽  
Endothelial Progenitor ◽  
And Function

Background Critical limb ischemia (CLI) is characterized by obstruction of lower extremity arteries and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and a dysfunctional BM environment contribute to impaired neovascularization in CLI. Methods Levels of primitive (CD34+ and CD133+) progenitors and CD34+KDR+ haemangioblastic EPC were analyzed using flow cytometry in peripheral blood (PB) and BM from 101 CLI patients in the JUVENTAS trial ( NCT00371371 ) and healthy controls (n=37 and n=12 for PB and BM, respectively). Endothelial damage markers (sE-selectin, sICAM-1, sVCAM-1, thrombomodulin) and PB levels of progenitor cell mobilizing (VEGF, SDF-1α, SCF, G-CSF) and inflammatory (IL-6, IL-8, IP-10) factors were assessed by ELISA and multiplex. Levels and activity of the EPC mobilizing protease MMP-9 were assessed in BM plasma by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured from PB, and CAC paracrine function was assessed. Results Endothelial damage markers were higher in CLI ( p< 0.01). PB levels of VEGF, SDF-1α, SCF, G-CSF ( p< 0.05) and of IL-6, IL-8 and IP-10 were higher in CLI ( p< 0.05). Circulating EPC and CD133+ cells and BM CD34+ cells were significantly lower in CLI (all p <0.05), BM levels and activity of MMP-9 were lower in CLI (both p< 0.01). Multivariate regression analysis showed an inverse association between IL-6 levels and BM CD34+ cell levels ( p= 0.007). CAC outgrowth did not differ significantly between CLI patients and healthy controls ( p= 0.137), however CAC from CLI patients had profoundly reduced migration stimulating potential ( p< 0.0001). Conclusion CLI patients have reduced levels of circulating EPC despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34+ cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. Our data suggest that reduced levels and function of circulating progenitor cells and BM dysfunction contribute to the defective neovascularization response in CLI.

Abstract 2876: Early Retention and Subsequent Engraftment of Transplanted Progenitor Cells is Improved by Delivery within Collagen Matrix

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yan Zhang ◽  
Marc Lamoureux ◽  
Stephanie Thorn ◽  
Vincent Chan ◽  
Joel Price ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Progenitor Cells ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Collagen Matrix ◽  
Cell Labeling ◽  
Type I ◽  
The Matrix ◽  
Early Retention

Background: To investigate the mechanisms involved in the potentiation of cell therapy by delivery matrices, we evaluated the retention and engraftment of transplanted human circulating progenitor cells (CPCs) injected in a collagen matrix by using in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and immunohistochemistry. Methods: CPCs were labeled with 18 F-FDG and injected with or without a collagen type I-based matrix in the ischemic hindlimb muscle (IM) of rats (2x10 6 cells; n=15/group). Localization of cells was acquired by PET imaging (15 min) at 150 min post-injection. In addition, radionuclide biodistribution, immunofluorescence, and immunohistochemical examination of transplanted CPCs were performed at up to 14 days. Results: Cell labeling efficiency was CPC-concentration dependent (r=0.61, p <0.001), but not 18 F-FDG-dose dependent. Labeled CPCs exhibited excellent short-term stability and viability. Persistence of 18 F-FDG radioactivity in cells was markedly greater than non-specific retention in the matrix. Wholebody (WB) PET images revealed better CPC retention in the IM and less non-specific leakage to other tissues when CPCs were delivered within the matrix (IM/WB retention ratio of 43.9±8.2%), compared to cells injected alone (22.3±10.4%; p =0.040) and to 18 F-FDG injected with or without the matrix (9.7±5.5% and 11.0±5.5%, respectively; p <0.005). Radioactivity biodistribution confirmed that accumulation was increased (by 92.5%; p =0.024) in the IM and reduced (by 1.1 to 23.8%; p <0.05) in non-specific tissues when cells were injected within the matrix, compared to cells injected alone. Anti-human mitochondria staining showed increased cell retention in the IM with use of matrices (3.0±2.1%) versus cells only (1.9±0.8%; p =0.048). At 14 days the number of CD31 + transplanted human cells was greater (1.6±0.1%) when injected within the matrix than injected alone (0.7±0.1%; p =0.004). Conclusions: Collagen-based delivery matrices improve the early retention of transplanted CPCs, which in turn favors subsequent cell engraftment in the ischemic tissue. This mechanism conferred by the matrix has potential implications for the optimization of cell therapy at the early stages after cell delivery.

Basement membrane and interstitial matrix components form separate matrices in heterokaryons of PYS-2 cells and fibroblasts

1993 ◽  
Vol 104 (1) ◽  
pp. 59-68
Author(s):  
P. Laurila ◽  
I. Leivo
Keyword(s):  
Basement Membrane ◽  
Matrix Protein ◽  
Spatial Organization ◽  
Gene Dosage ◽  
Parental Cell ◽  
Cell Nuclei ◽  
Fibronectin Matrix ◽  
Basement Membrane Protein ◽  
The Matrix

In order to gain further understanding of the spatial organization of interstitial and basement membrane matrices, we studied the expression of the interstitial matrix protein, fibronectin, and the basement membrane protein, laminin, in heterokaryons formed by the fusion of normal fibroblasts and teratocarcinoma-derived epithelial PYS-2 cells. These heterokaryons showed various distributions of the matrix proteins depending on the proportions of the different parental cell nuclei within the cytoplasm of the cell. Heterokaryons containing equal numbers of fibroblast and PYS-2 cell nuclei showed an abundant laminin matrix subcellularly and only minor amounts of fibronectin matrix at the periphery of the cells. Similar results were obtained in heterokaryons containing an excess of epithelial cell nuclei. In heterokaryons containing an excess of fibroblast nuclei, on the other hand, laminin matrix was reduced and a fibrillar fibronectin matrix was seen also on top of the cell body. The results suggest a gene dosage-type of effect on the expression of these proteins. Furthermore, extracellular laminin and fibronectin matrices did not codistribute around the heterokaryons but the two proteins were assembled into separate structures. The lack of codistribution of fibronectin and laminin matrices in heterokaryons suggests that the molecular interactions, which determine the assembly of basement membrane and interstitial matrices in these cells are highly type-specific. Similar mechanisms may also operate in the assembly of extracellular matrices in vivo.

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