Contribution of the fibrinolytic pathway to hematopoietic regeneration

Hematopoietic stem cells (HSCs) can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression, infections, and allergic reactions), thereby ensuring a well‐regulated supply of mature and immature hematopoietic cells within the circulation and prompt adjustment of blood cell levels within normal ranges. The recovery of tissues and organs from hematopoietic stress (e.g., myelosuppression or ionizing irradiation) is dependent on two cell types: resident HSCs which repopulate the bone marrow (BM) cavity, and stromal cells. BM regeneration critically depends on the release of soluble factors from cells such as stromal cells, a process regulated by proteases. Two proteolytic systems, the fibrinolytic system and the matrix metalloproteinases (MMPs), have recently been shown to be involved in this process (Heissig B, 2007, Cell Stem Cell 1: 658–670). The plasminogen/plasmin system is mostly recognized for its fibrinolytic activity, but it is also involved in processes such as cell invasion, chemotaxis, growth factor activity modulation, and tissue remodeling. This review focuses on the role of plasmin and its activators as key players in controlling the hematopoietic stress response after myelosuppression (hematopoietic regeneration). Aspects of plasmin regulation, especially regulation of its ability to activate MMPs and the functional consequences of this enzyme activation, such as plasmin‐mediated release of biologically relevant cytokines from the matrix and cell surfaces, will be discussed. J. Cell. Physiol. 221: 521–525, 2009. © 2009 Wiley‐Liss, Inc.     

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.     

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.     

Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.     

Plasmin-mediated Proteolysis Is Required for Hepatocyte Growth Factor Activation during Liver Repair

The physiological relevance of the activation of hepatocyte growth factor (Hgf) by the plasminogen (Plg) system of proteases and its contribution to tissue repair are largely undefined. Here, we investigated whether the defective liver repair in mice lacking Plg is due to impaired activation of Hgf. Loss of Plg in vivo suppressed Hgf activation and signaling through its Met tyrosine kinase receptor. Without Plg, hepatocytes were unresponsive to Hgf-induced proliferation and migration, with a more pronounced impairment in hepatocyte movement within the hepatic environment. Most notably, circumventing the defect in proteolytic activation of Hgf by the downstream expression of an activated Met receptor corrected the functional deficits and improved liver repair in Plg-deficient mice. These findings support a fibrinolysis-unrelated role for Plg in modulating cell proliferation and migration by activation of Hgf.Tissue repair requires a prompt proliferative response in concert with the timely reorganization of the extracellular matrix. Each one of these processes can be disrupted by the loss of individual growth factors or proteases, but the precise regulatory relationship between these molecules in supporting tissue repair is not fully understood. Multiple in vitro studies have inferred that proteases in the plasminogen (Plg)² activation system may be important in the proteolytic activation of the hepatocyte growth factor (Hgf) (1–4), the ligand for the Met tyrosine kinase receptor that exerts potent mitogenic and motogenic properties to mesenchymal and epithelial cells. This concept is made even more attractive by the fact that Hgf is structurally related to Plg, with multiple kringle domains and a catalytically inactive serine protease-like domain. However, the physiological relevance of Plg to Hgf activation and Hgf-related reparative processes are controversial and effectively unexplored in vivo.We previously reported that a genetically imposed loss of circulating Plg severely impairs clearance of necrotic cells and the repopulation of injured zones by newly formed cells but without compromising the general hepatic proliferative response (5). Despite the indisputable role of Plg in fibrin clearance (6), complementary studies in mice with no capacity for fibrin deposition have shown that the loss of fibrinolytic function alone in Plg-deficient mice cannot account for the impediment in tissue repair (5). Multiple nonfibrin targets of plasmin-mediated proteolysis are known (e.g. serine and metalloprotease zymogens, and extracellular matrix glycoproteins, latent growth factors), and it is feasible that they may contribute to the focal clearance of necrotic tissue. However, based on recent findings pointing to a strikingly similar defect in hepatic repair in mice lacking Plg or a conditional loss of Met (7), an attractive hypothesis emerged that the Plg activation system supports physiological liver repair by activation of the Met ligand, Hgf. Testing this hypothesis, we found that the loss of Plg impairs Hgf activation, suppresses Met phosphorylation and signaling, and prevents Hgf-induced migration of hepatocytes. Most notably, consistent with a physiologically relevant contribution of Plg to Hgf-Met signaling, the expression of an autophosphorylated Met largely corrected the defective repair in Plg-deficient livers.     

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.     

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.     

Inhibition of hematopoietic recovery from radiation-induced myelosuppression by natural killer cells

We have examined the role of natural killer (NK) cells in situ in the recovery of marrow hematopoiesis in B6D2F1 mice receiving various doses of total-body irradiation (TBI) as a well-characterized model for treatment-induced myelosuppression. Applying an in situ cytotoxic approach for ablating NK 1.1 cells, we have demonstrated that NK 1.1 cells differentially inhibit the recovery of hematopoietic stem cells (CFU-S) and their progenitor cells committed to granulocyte-macrophage differentiation from a sublethal dose of TBI (9 Gy) while not affecting the recovery of progenitor cells committed to either erythroid or megakaryocyte differentiation from TBI. However, recoveries of CFU-S and progenitor cells were unaffected by the ablation of NK cells prior to a moderate dose of TBI (2 Gy). These findings provide in situ evidence that NK cells are potential inhibitors of hematopoietic recovery from treatment-induced myelosuppression.     

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.     

Lactational competence and involution of the mouse mammary gland require plasminogen

Urokinase-type plasminogen activator expression is induced in the mouse mammary gland during development and post-lactational involution. We now show that primiparous plasminogen-deficient (Plg(-/-)) mice have seriously compromised mammary gland development and involution. All mammary glands were underdeveloped and one-quarter of the mice failed to lactate. Although the glands from lactating Plg(-/-) mice were initially smaller, they failed to involute after weaning, and in most cases they failed to support a second litter. Alveolar regression was markedly reduced and a fibrotic stroma accumulated in Plg(-/-) mice. Nevertheless, urokinase and matrix metalloproteinases (MMPs) were upregulated normally in involuting glands of Plg(-/-) mice, and fibrin did not accumulate in the glands. Heterozygous Plg(+/-) mice exhibited haploinsufficiency, with a definite, but less severe mammary phenotype. These data demonstrate a critical, dose-dependent requirement for Plg in lactational differentiation and mammary gland remodeling during involution.     

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.     

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

PbsP,a cell wall‐anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine‐rich region, and a LPXTG cell wall‐anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface‐bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non‐CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.