Sylwia Borkowska

Ceramide-1-Phosphate Regulates Migration of Multipotent Stromal Cells and Endothelial Progenitor Cells—Implications for Tissue Regeneration†‡§

Ceramide‐1‐phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an antiapoptotic molecule released from cells that are damaged and “leaky.” As reported recently, C1P promotes migration of hematopoietic cells. In this article, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for the first time that C1P is upregulated in damaged tissues and chemoattracts bone marrow (BM)‐derived multipotent stromal cells, endothelial progenitor cells, and very small embryonic‐like stem cells. Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal cell‐derived factor‐1 from BM‐derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization. STEM CELLS2013;31:500–510

Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs).

The role of blood proteinases in the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not well understood. As previously reported, activation of the complement cascade (ComC) and cleavage of C5 by C5 convertase are enabling events in the release of C5a that plays a crucial role in the egress of HSPCs from bone marrow (BM) into peripheral blood (PB) and explains why C5-deficient mice are poor mobilizers. Here we provide evidence that during granulocyte colony-stimulating factor- and AMD3100-induced mobilization, not only the ComC but also two other evolutionarily ancient proteolytic enzyme cascades, the coagulation cascade (CoaC) and the fibrynolytic cascade (FibC), become activated. Activation of all three cascades was measured by generation of C5a, decrease in prothrombin time and activated partial thromboplastin time as well as an increase in the concentrations of plasmin/antiplasmin and thrombin/antithrombin. More importantly, the CoaC and FibC, by generating thrombin and plasmin, respectively, provide C5 convertase activity, explaining why mobilization of HSPCs in C3-deficient mice, which do not generate ComC-generated C5a convertase, is not impaired. Our observations shed more light on how the CoaC and FibC modulate stem cell mobilization and may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, as it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (for example, infections, tissue/organ damage or strenuous exercise) and show a circadian rhythm of activation, they must be involved in both stress-induced and circadian changes in HSPC trafficking in PB.

Hematopoietic Stem/Progenitor Cells Express Several Functional Sex Hormone Receptors—Novel Evidence for a Potential Developmental Link Between Hematopoiesis and Primordial Germ Cells

Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45(-) very small embryonic-like stem cells (VSELs) may become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy.

The role of sphingosine-1 phosphate and ceramide-1 phosphate in trafficking of normal stem cells and cancer cells

Introduction: A common feature of many types of cells is their responsiveness to chemotactic gradients of factors for which they express the corresponding receptors. The most studied chemoattractants so far are peptide-based growth factors and a family of cytokines endowed with strong chemotactic properties, called chemokines. However, additional evidence has accumulated that, in addition to these peptide-based chemoattractants, an important role in cell migration is played by bioactive lipids. Areas covered: Solid evidence has accumulated that two bioactive phosphorylated sphingolipids that are derivatives of sphingolipid metabolism, namely sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are potent chemoattractants for a variety of cells. In this review, we will discuss the effect of these two phosphorylated sphingolipids on the trafficking of normal and malignant cells, and, in particular, we will focus on their role in trafficking of normal hematopoietic stem/progenitor cells. Unlike other mediators, S1P under steady-state conditions maintain a steep gradient between interstitial fluid and peripheral blood and lymph across the endothelial barrier, which is important in the egress of cells from bone marrow. Both S1P and C1P may be upregulated in damaged tissues, which may result in reversal of this gradient. Expert opinion: S1P and C1P are important regulators of the trafficking of normal and malignant cells, and modification of their biological effects will have important applications in optimizing stem cell mobilization and homing, tissue organ/regeneration, and preventing cancer metastasis.

Human haematopoietic stem/progenitor cells express several functional sex hormone receptors.

Evidence has accumulated that murine haematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs. It has also been reported that human HSPCs, like their murine counterparts, respond to certain SexHs (e.g. androgens). However, to better address the effects of SexHs, particularly pituitary SexHs, on human haematopoiesis, we tested for expression of receptors for pituitary SexHs, including follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, oestrogens, and androgen, on HSPCs purified from human umbilical cord blood (UCB) and peripheral blood (PB). We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB‐derived, CD45− very small embryonic‐like stem cells (VSELs) become specified into CD45+ HSPCs, we also evaluated the expression of pituitary and gonadal SexH receptors on these cells. We report for the first time that human HSPCs and VSELs, like their murine counterparts, express pituitary and gonadal SexH receptors at the mRNA and protein levels. Most importantly, SexH if added to suboptimal doses of haematopoietic cytokines and growth factors enhance clonogenic growth of human HSPCs as well as directly stimulate proliferation of MSCs.

Complement Component 3 is Necessary to Preserve Myocardium and Myocardial Function in Chronic Myocardial Infarction

Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post‐MI. Morphometric analysis 28 days post‐MI showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c‐kitpos cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67pos CSPCs and decreased formation of new BrdUpos/α‐sarcomeric actinpos myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post‐MI in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post‐MI. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function. Stem Cells 2014;32:2502–2515

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment

The concept that bone marrow (BM)‐derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca‐1+Lin−CD45− cells known as very small embryonic/epiblast‐like stem cells (VSELs) that express several markers of pluripotency such as Oct‐4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPKp42/44 and AKTser473. Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Evidence of a Pivotal Role for the Distal Part of the Complement Cascade in the Diurnal Release of Hematopoietic Stem Cells Into Peripheral Blood.

We found that diurnal activation of the three evolutionarily ancient proteolytic cascades in peripheral blood (PB), namely, the complement, coagulation, and fibrinolytic cascades, late at night or in the early morning hours, precedes the diurnal release of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into PB in wild-type mice. Moreover, activation of the distal part of the complement cascade (ComC), involving cleavage of the fifth component (C5), seems to play a crucial role in pharmacological mobilization of HSPCs. In order to shed more light on the role of diurnal rhythms in the egress of HSPCs, we studied diurnal changes in the number of circulating HSPCs in C5-deficient mice and did not observe diurnal changes in the number of these cells circulating in PB in C5-/- animals. Based on this finding, we conclude that activation of the distal part of the ComC, C5 cleavage, and release of C5a and desArgC5a are required in executing the diurnal release of HSPCs from BM into PB. Moreover, the fact that C5-/- mice still displayed normal activation of the coagulation and fibrinolytic cascades indicates that, of all the proteolytic cascades, the ComC is the dominant player regulating diurnal egress of HSPCs.