Bioactive Sphingolipids, Complement Cascade, and Free Hemoglobin Levels in Stable Coronary Artery Disease and Acute Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) and coronary artery bypass graft (CABG) surgery are associated with a pathogen-free inflammatory response (sterile inflammation). Complement cascade (CC) and bioactive sphingolipids (BS) are postulated to be involved in this process. AIM: The aim of this study was to evaluate plasma levels of CC cleavage fragments (C3a, C5a, and C5b9), sphingosine (SP), sphingosine-1-phosphate (S1P), and free hemoglobin (fHb) in AMI patients treated with primary percutaneous coronary intervention (pPCI) and stable coronary artery disease (SCAD) undergoing CABG. PATIENTS AND METHODS: The study enrolled 37 subjects (27 male) including 22 AMI patients, 7 CABG patients, and 8 healthy individuals as the control group (CTRL). In the AMI group, blood samples were collected at 5 time points (admission to hospital, 6, 12, 24, and 48 hours post pPCI) and 4 time points in the CABG group (6, 12, 24, and 48 hours post operation). SP and S1P concentrations were measured by high-performance liquid chromatography (HPLC). Analysis of C3a, C5a, and C5b9 levels was carried out using high-sensitivity ELISA and free hemoglobin by spectrophotometry. RESULTS: The plasma levels of CC cleavage fragments (C3a and C5b9) were significantly higher, while those of SP and S1P were lower in patients undergoing CABG surgery in comparison to the AMI group. In both groups, levels of CC factors showed no significant changes within 48 hours of follow-up. Conversely, SP and S1P levels gradually decreased throughout 48 hours in the AMI group but remained stable after CABG. Moreover, the fHb concentration was significantly higher after 24 and 48 hours post pPCI compared to the corresponding postoperative time points. Additionally, the fHb concentrations increased between 12 and 48 hours after PCI in patients with AMI. CONCLUSIONS: Inflammatory response after AMI and CABG differed regarding the release of sphingolipids, free hemoglobin, and complement cascade cleavage fragments.

Evidence that a lipolytic enzyme--hematopoietic-specific phospholipase C-ß2--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 α4ß1-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-ß2 (PLC-ß2) 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-ß2-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 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.

Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate.

The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2-H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation.

Parental imprinting regulates insulin-like growth factor signaling: a Rosetta Stone for understanding the biology of pluripotent stem cells, aging and cancerogenesis.

In recent years, solid evidence has accumulated that insulin-like growth factor-1 (IGF-1) and 2 (IGF-2) regulate many biological processes in normal and malignant cells. Recently, more light has been shed on the epigenetic mechanisms regulating expression of genes involved in IGF signaling (IFS) and it has become evident that these mechanisms are crucial for initiation of embryogenesis, maintaining the quiescence of pluripotent stem cells deposited in adult tissues (for example, very-small embryonic-like stem cells), the aging process, and the malignant transformation of cells. The expression of several genes involved in IFS is regulated at the epigenetic level by imprinting/methylation within differentially methylated regions (DMRs), which regulate their expression from paternal or maternal chromosomes. The most important role in the regulation of IFS gene expression is played by the Igf-2-H19 locus, which encodes the autocrine/paracrine mitogen IGF-2 and the H19 gene, which gives rise to a non-coding RNA precursor of several microRNAs that negatively affect cell proliferation. Among these, miR-675 has recently been demonstrated to downregulate expression of the IGF-1 receptor. The proper imprinting of DMRs at the Igf-2-H19 locus, with methylation of the paternal chromosome and a lack of methylation on the maternal chromosome, regulates expression of these genes so that Igf-2 is transcribed only from the paternal chromosome and H19 (including miR-675) only from the maternal chromosome. In this review, we will discuss the relevance of (i) proper somatic imprinting, (ii) erasure of imprinting and (iii) loss of imprinting within the DMRs at the Igf-2-H19 locus to the expression of genes involved in IFS, and the consequences of these alternative patterns of imprinting for stem cell biology.

Pluripotent and multipotent stem cells in adult tissues.

One of the most intriguing questions in stem cell biology is whether pluripotent stem cells exist in adult tissues. Several groups of investigators employing i) various isolation protocols, ii) detection of surface markers, and iii) experimental in vitro and in vivo models, have reported the presence of cells that possess a pluripotent character in adult tissues. Such cells were assigned various operational abbreviations and names in the literature that added confusion to the field and raised the basic question of whether these are truly distinct or overlapping populations of the same primitive stem cells. Unfortunately, these cells were never characterized side-by-side to address this important issue. Nevertheless, taking into consideration their common features described in the literature, it is very likely that various investigators have described overlapping populations of developmentally early stem cells that are closely related. These different populations of stem cells will be reviewed in this paper.

Pivotal role of paracrine effects in stem cell therapies in regenerative medicine: can we translate stem cell-secreted paracrine factors and microvesicles into better therapeutic strategies?

Although regenerative medicine is searching for pluripotent stem cells that could be employed for therapy, various types of more differentiated adult stem and progenitor cells are in meantime being employed in clinical trials to regenerate damaged organs (for example, heart, kidney or neural tissues). It is striking that, for a variety of these cells, the currently observed final outcomes of cellular therapies are often similar. This fact and the lack of convincing documentation for donor-recipient chimerism in treated tissues in most of the studies indicates that a mechanism other than transdifferentiation of cells infused systemically into peripheral blood or injected directly into damaged organs may have an important role. In this review, we will discuss the role of (i) growth factors, cytokines, chemokines and bioactive lipids and (ii) microvesicles (MVs) released from cells employed as cellular therapeutics in regenerative medicine. In particular, stem cells are a rich source of these soluble factors and MVs released from their surface may deliver RNA and microRNA into damaged organs. Based on these phenomena, we suggest that paracrine effects make major contributions in most of the currently reported positive results in clinical trials employing adult stem cells. We will also present possibilities for how these paracrine mechanisms could be exploited in regenerative medicine to achieve better therapeutic outcomes. This approach may yield critical improvements in current cell therapies before true pluripotent stem cells isolated in sufficient quantities from adult tissues and successfully expanded ex vivo will be employed in the clinic.

A novel perspective on stem cell homing and mobilization: review on bioactive lipids as potent chemoattractants and cationic peptides as underappreciated modulators of responsiveness to SDF-1 gradients.

Hematopoietic stem progenitor cells (HSPCs) respond robustly to α-chemokine stromal-derived factor-1 (SDF-1) gradients, and blockage of CXCR4, a seven-transmembrane-spanning G(αI)-protein-coupled SDF-1 receptor, mobilizes HSPCs into peripheral blood. Although the SDF-1-CXCR4 axis has an unquestionably important role in the retention of HSPCs in bone marrow (BM), new evidence shows that, in addition to SDF-1, the migration of HSPCs is directed by gradients of the bioactive lipids sphingosine-1 phosphate and ceramide-1 phosphate. Furthermore, the SDF-1 gradient may be positively primed/modulated by cationic peptides (C3a anaphylatoxin and cathelicidin) and, as previously demonstrated, HSPCs respond robustly even to very low SDF-1 gradients in the presence of priming factors. In this review, we discuss the role of bioactive lipids in stem cell trafficking and the consequences of HSPC priming by cationic peptides. Together, these phenomena support a picture in which the SDF-1-CXCR4 axis modulates homing, BM retention and mobilization of HSPCs in a more complex way than previously envisioned.

Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b-C9 as homing factors.

We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

The bone marrow-expressed antimicrobial cationic peptide LL-37 enhances the responsiveness of hematopoietic stem progenitor cells to an SDF-1 gradient and accelerates their engraftment after transplantation.

We report that the bone marrow (BM) stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases the responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increasing their (i) adhesiveness, (ii) SDF-1-mediated actin polymerization and (iii) MAPK(p42/44) phosphorylation. Mice transplanted with BM cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ∼3-5 days compared with mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on the incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as an ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited.

Hematopoietic differentiation of umbilical cord blood-derived very small embryonic/epiblast-like stem cells.

A population of CD133(+)Lin(-)CD45(-) very small embryonic/epiblast-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). To speed up isolation of these cells, we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45(-)/GlyA(-)/CD133(+)/ALDH(high) and CD45(-)/GlyA(-)/CD133(+)/ALDH(low) cells, which are enriched for VSELs, and CD45(+)/GlyA /CD133(+)/ALDH(high) and CD45(+)/GlyA(-)/CD133(+)/ALDH(low) cells, which are enriched for hematopoietic stem/progenitor cells (HSPCs). Although freshly isolated CD45(-) VSELs did not grow hematopoietic colonies, the same cells, when activated/expanded over OP9 stromal support, acquired hematopoietic potential and grew colonies composed of CD45(+) hematopoietic cells in methylcellulose cultures. We also observed that CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs grew colonies earlier than CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs, which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility, real-time polymerase chain reaction analysis confirmed that, whereas freshly isolated CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs express more hematopoietic transcripts (for example, c-myb), CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs exhibit higher levels of pluripotent stem cell markers (for example, Oct-4). More importantly, hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated non-obese diabetic/severe combined immunodeficiency mice 4-6 weeks after transplantation. Overall, our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB.

Stem cells for neural regeneration--a potential application of very small embryonic-like stem cells.

The goal of regenerative medicine is to ameliorate irreversible destruction of brain tissue by harnessing the power of stem cells in the process of neurogenesis. Several types of stem cells, including mesenchymal stem cells, hematopoietic stem cells, as well as neural cells differentiated from embryonic stem cell lines, have been proposed as potential therapeutic vehicles. In this review paper we will discuss a perspective of stem cell therapies for neurological disorders with special emphasis on potential application of cells isolated from adult tissues. In support of this our group found that murine bone marrow contains a mobile population of Oct-4+CXCR4+SSEA-1+Sca-1+lin⁻CD45⁻ very small embryonic-like stem cells (VSELs) that are mobilized into peripheral blood in a murine stroke model. The number of these cells in circulation increases also after pharmacological mobilization by administration of granulocyte colony stimulating factor (G-CSF). Recently we found that VSELs are present in various non-hematopoietic adult organs and, interestingly, our data indicate that the brain contains a high number of cells that display the VSEL phenotype. Based on our published data both in human and mice we postulate that VSELs are a mobile population of epiblast/germ line-derived stem cells and play an important role as an organ-residing reserve population of pluripotent stem cells that give rise to stem cells committed to particular organs and tissues--including neural tissue. In conclusion human VSELs could be potentially harnessed in regenerative medicine as a source of stem cells for neurogenesis.

High-protein diet during gestation and lactation affects mammary gland mRNA abundance, milk composition and pre-weaning litter growth in mice.

We evaluated the effect of a high-protein diet (HP) on pregnancy, lactational and rearing success in mice. At the time of mating, females were randomly assigned to isoenergetic diets with HP (40% w/w) or control protein levels (C; 20%). After parturition, half of the dams were fed the other diet throughout lactation resulting in four dietary groups: CC (C diet during gestation and lactation), CHP (C diet during gestation and HP diet during lactation), HPC (HP diet during gestation and C diet during lactation) and HPHP (HP diet during gestation and lactation). Maternal and offspring body mass was monitored. Measurements of maternal mammary gland (MG), kidney and abdominal fat pad masses, MG histology and MG mRNA abundance, as well as milk composition were taken at selected time points. HP diet decreased abdominal fat and increased kidney mass of lactating dams. Litter mass at birth was lower in HP than in C dams (14.8 v. 16.8 g). Dams fed an HP diet during lactation showed 5% less food intake (10.4 v. 10.9 g/day) and lower body and MG mass. On day 14 of lactation, the proportion of MG parenchyma was lower in dams fed an HP diet during gestation as compared to dams fed a C diet (64.8% v. 75.8%). Abundance of MG α-lactalbumin, ß-casein, whey acidic protein, xanthine oxidoreductase mRNA at mid-lactation was decreased in all groups receiving an HP diet either during gestation and/or lactation. Milk lactose content was lower in dams fed an HP diet during lactation compared to dams fed a C diet (1.6% v. 2.0%). On days 14, 18 and 21 of lactation total litter mass was lower in litters of dams fed an HP diet during lactation, and the pups' relative kidney mass was greater than in litters suckled by dams receiving a C diet. These findings indicate that excess protein intake in reproducing mice has adverse effects on offspring early in their postnatal growth as a consequence of impaired lactational function.

Innate immunity as orchestrator of stem cell mobilization.

Hematopoietic stem and progenitor cells (HSPCs), as well as other types of stem cells, circulate under steady-state conditions at detectable levels in peripheral blood (PB), with their numbers increasing in response to stress, inflammation and tissue/organ injury. This mobilization process may be envisioned as a danger-sensing response mechanism triggered by hypoxia or mechanical or infection-induced tissue damage that recruits into PB different types of stem cells that have a role in immune surveillance and organ/tissue regeneration. Mobilization is also significantly enhanced by the administration of pharmacological agents, which has been exploited in hematological transplantology as a means to obtain HSPCs for hematopoietic reconstitution. In this review we will present mounting evidence that innate immunity orchestrates this evolutionarily conserved mechanism of HSPC mobilization.

Molecular signature of adult bone marrow-purified very small embryonic-like stem cells supports their developmental epiblast/germ line origin.

We postulated that Oct4(+)SSEA-1(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) isolated from adult bone marrow (BM) could be a reserve population for tissue-committed stem cells. The aim of this study was to elucidate the developmental origin of these cells. We report that during embryogenesis, VSELs are enriched in embryonic day (E)12.5 murine fetal livers (FLs) and subsequently follow the developmental route of hematopoietic stem cells (H)SCs to colonize BM. Molecular analysis of purified VSELs revealed that both FL-derived VSELs and their adult BM-derived counterparts express: (i) several epiblast/primordial germ cell (PGC) markers; (ii) migrating PGC-like epigenetic reprogramming profiles of Oct4, Nanog and Stella loci; as well as (iii) a unique pattern of genomic imprinting. Thus, these data suggest that VSELs may originate from epiblast/migrating PGC-like cells and, in spite of the expression of pluripotent stem cell markers, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation.

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.