Erythropoiesis PDF

ERYTHROPOIESIS PhD. MD. Marsela Haruni  Learning Objectives for Erythropoiesis ▪ Define Erythropoiesis: Understand and explain the process of erythropoiesis and its role in hematopoiesis. ▪ Identify Key Stages: Describe the stages of erythrocyte development, from hemat...

ERYTHROPOIESIS PhD. MD. Marsela Haruni  Learning Objectives for Erythropoiesis ▪ Define Erythropoiesis: Understand and explain the process of erythropoiesis and its role in hematopoiesis. ▪ Identify Key Stages: Describe the stages of erythrocyte development, from hematopoietic stem cells to mature erythrocytes, including the morphological changes that occur at each stage. ▪ Understand Regulatory Factors: Analyze the hormonal and microenvironmental factors that regulate erythropoiesis, particularly the role of erythropoietin (EPO).  CBC With Differential/Platelet What does a Complete Blood Count measure? What are the components of a CBC? How is a CBC performed? What do the results of a CBC tell us about health condition? What do the terms "differential" and "platelet" mean in relation to a CBC?  A complete blood count (CBC) is one of the most commonly ordered laboratory test panels. It provides relative numbers and calculations obtained from the cells (erythrocytes and leukocytes) and formed elements (thrombocytes) in the blood sample. These calculations are usually performed by automated blood cell counters that analyze different components of blood using the principle of flow cytometry design. In preparation for counting, the blood sample is diluted in a suspension fluid. As a thin stream of fluid with suspended cells flows through the narrow tubing of the cell counter, the light detector and electrical impedance sensor identify different cell types based on their size and electrical resistance. Data obtained from automatic blood analyzers are usually very accurate. Computer-assisted blood cell analysis systems use cameras and image-processing technologies to automatically count and analyze cells.  Complete Blood Count (CBC) measures several key components of blood, including: 1.Red Blood Cells (RBCs): These carry oxygen from the lungs to the rest of the body and help remove carbon dioxide. 2.Hemoglobin (Hb): The protein in red blood cells that carries oxygen. 3.Hematocrit (Hct): The percentage of blood volume that is made up of red blood cells. 4.White Blood Cells (WBCs): These are part of the immune system and help fight infections. 5.White Blood Cell Differential: The major types of WBCs reported are neutrophils, eosinophils, basophils, lymphocytes, and monocytes. 6.Platelets: These are small cell fragments that help with blood clotting. Together, these measurements provide important insights into your overall health and help detect various medical conditions. Where do these different blood cells come from? They are all descendants of a single precursor cell type known as the pluripotent hematopoietic stem cell. This cell type is found primarily in bone marrow, a soft tissue that fills the hollow center of bones. Pluripotent stem cells have the remarkable ability to develop into many different cell types. As they specialize, they narrow their possible fates. First, they become uncommitted stem cells, then progenitor cells that are committed to developing into one or perhaps two cell types. Progenitor cells differentiate into red blood cells, lymphocytes, other white blood cells, and megakaryocytes, the parent cells of platelets. It is estimated that only about one out of every 100,000 cells in the bone marrow is an uncommitted stem cell, making it difficult to isolate and study these cells.  ERYTHROPOIESIS The production of erythrocytes is a tightly regulated process. During steady state hematopoiesis, approximately 1010 red blood cells are produced per hour in the bone marrow to maintain the hemoglobin level within fairly narrow limits. Production can be rapidly increased in the setting of ongoing blood loss or hemolysis. Erythropoiesis begins with the differentiation of multipotent hematopoietic stem cells (HSC) into the most primitive erythroid progenitors. In the classical hematopoietic hierarchy, HSC progeny can differentiate into a common myeloid progenitor (CMP), which in turn differentiates into the common megakaryocyte-erythroid progenitor cell (MEP). Studies in humans and mouse models using transplantation and single-cell lineage tracing have shown that the MEPs may derive directly from HSCs. MEPs give rise to committed megakaryocyte and erythroid progenitor cells; The latter subsequently follow a differentiation program that culminates in the emergence of mature erythrocytes. Iron deficiency is associated not only with anemia but frequently with thrombocytosis as well and is associated with a two-fold increase in thrombotic risk. Iron metabolism is an evolutionarily highly conserved process. Data from mouse models show that an iron-deficient diet or knockout of the gene that is altered in iron-refractory iron deficiency anemia (IRIDA), TMPRSS6, have thrombocytosis. The mechanism was found to involve increased flux of progenitor cells through MEPs, with evidence for attenuation of extracellular signal-regulated kinase (ERK) phosphorylation. This mechanism illustrates an example of extracellular influences on progenitor cell fate.  ERYTHROID PROGENITOR CELLS The erythroid progenitor compartment of the bone marrow is invisible upon inspection by light microscopy. These committed single lineage progenitors are derived from the stochastic differentiation of bipotential or multipotential progenitors, which emerged from a tiny population of stem cells. The two sets of erythroid progenitors described below, burst-forming units of the erythroid lineage and colony-forming units of the erythrocyte lineage (BFU-E and CFU-E), cannot be identified by specific morphological features, although they have been purified and analyzed using flow cytometric methods. Evidence suggests that all hematopoietic progenitors or stem cells resemble lymphoblasts [7-9].  Erythroid burst-forming unit In humans, the most primitive single lineage committed erythroid progenitor is the erythroid burst-forming unit (BFU-E). These cellular clusters are so named because they have the following characteristics in semisolid in vitro cultures: The colonies have a burst-like morphology. In response to the combination of EPO and one of SCF, IL-3, or GM-CSF, the progeny of the first few cellular divisions are motile and form subpopulations of erythroid colony-forming units (CFU-E). Each of these units subsequently forms a large colony of proerythroblasts, which mature into erythroblasts and a few enucleated erythrocytes. The entire process requires approximately two weeks in vitro.  Erythroid colony-forming unit Bone marrow also contains the more mature erythroid colony-forming unit (CFU-E) that, under the influence of EPO, form small colonies of erythroblasts in seven days of culture. PRECURSORS AND MATURE CELLS The erythroid precursor or erythroblast pool represents about one-third of the marrow cell population in the normal child (above the age of three) and the adult. Proerythroblasts are the earliest recognizable forms. Erythroid precursors in the bone marrow Erythroid maturation These cells divide and mature through basophilic, polychromatic, and orthochromatic normoblast cells to form the reticulocyte and finally to the circulating mature erythrocyte. This process involves a reduction in cell size, nuclear condensation and extrusion, and hemoglobin accumulation. On average, each proerythroblast can form approximately eight reticulocytes. The mean transit time from proerythroblast to the emergence of the reticulocyte into the circulation is approximately five days. In the circulation, the reticulocyte takes approximately one day to become the mature erythrocyte. With worsening anemia and increasing erythropoietin stimulation, bone marrow reticulocytes (left) leave the marrow at an earlier stage in their maturation. This prolongs the maturation time in the circulation from one day to as long as 2.5 days (right).  STROMA The bone marrow stroma consists of fibroblastoid cells, endothelial cells, and macrophages that comprise the hematopoietic microenvironment [16,17]. Interaction between receptors on erythrocyte precursors and elements of the stromal matrix are important determinants of erythroid maturation. Normal burst-forming units of the erythroid lineage (BFU-E) bind preferentially to a stromal fibroblast cell strain ; after binding, these cells proliferate and differentiate in the presence of EPO alone. Macrophages — Interactions between developing erythroid cells and macrophages are also important [21-26]. This is classically exemplified by the "erythroblastic island", composed of developing erythroblasts surrounding a central macrophage. These macrophages make physical contacts with developing erythroblasts, enabling signaling and the transfer of growth factors and nutrients (eg, iron) to these cells.  TRANSCRIPTION FACTORS Much has been learned about the essential roles of several transcription factors. Important factors that most likely act at the level of hematopoietic stem cells include TAL1/SCL, LMO2/RBTN2, and GATA2, while GATA1, FOG1 and EKLF are more important for erythropoiesis. TAL1/SCL — T-cell acute lymphocytic leukemia 1 (TAL1; also called stem cell leukemia [SCL]) transcription factor is expressed in leukemias (biphenotypic [lymphoid/myeloid] and T cells) [31,32], primitive hematopoietic progenitors, and more mature erythroid, mast, megakaryocyte, and endothelial cells [33,34]. Targeted disruption of this gene in mice leads to death in utero from the absence of blood formation. LMO2/RBTN2 — Another transcription factor implicated in T cell acute lymphoblastic leukemia is the LIM-only domain nuclear protein 2 (LMO2; also called rhombotin 2 [RBTN2]) [41,42]. Mice that lack this factor die in utero and have the same bloodless phenotype as animals that lack TAL1. GATA2 — GATA2 is highly expressed in progenitor cells. Overexpression of GATA2 in chicken erythroid progenitors leads to proliferation at the expense of differentiation. On the other hand, targeted disruption of the GATA2 gene leads to reduced primitive hematopoiesis in the yolk sac and embryonic death by day 10 to 11. GATA1 — GATA1 expression is limited to multipotent progenitors and to erythroid, megakaryocyte, mast, and eosinophil lineages [53-56]  GROWTH FACTORS The regulation of the proliferation and maturation of erythroid progenitors depends upon interaction with a number of growth factors. The availability of pure recombinant growth factors, enrichment of target progenitor cells, use of defined "serum-free" culture conditions, and the targeted disruption of factors and their receptors have provided insights into the role of these factors during hematopoiesis.  Erythropoietin EPO has long been recognized as the physiologic regulator of red cell production. It is produced in the kidney and the fetal liver in response to hypoxia or exposure to cobalt chloride. Erythropoietin (EPO) is essential for the terminal maturation of erythroid cells. Its major effect appears to be at the level of the CFU-E during adult erythropoiesis; CFU-E do not survive in vitro in the absence of EPO. Since the majority of CFU-E are cycling, their survival in the presence of EPO may be tightly linked to their proliferation and differentiation to mature erythrocytes. EPO also acts upon a subset of presumptive mature burst-forming units of the erythroid lineage (BFU-E), which requires EPO for survival and terminal maturation. A second subset of BFU-E, presumably less mature, survive EPO deprivation if other hematopoietic growth factors such as IL-3 or GM-CSF are present. Stem cell factor — Although stem cell factor (SCF) alone has no colony-forming ability, it has marked synergistic effects on BFU-E cultured in the presence of EPO [93-96]. SCF is crucial for the normal development of CFU-E. IL-3 and GM-CSF — Interleukin 3 (IL-3) and GM-CSF receptors share a common beta chain. Both cytokines enhance erythropoietin-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. Insulin and insulin-like growth factor 1 — Factors distinct from the classical colony-stimulating factors may positively regulate erythropoiesis, either directly or indirectly.  ERYTHROCYTES Erythrocytes are anucleate, biconcave discs. Erythrocytes or red blood cells (RBCs) are anucleate cells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave disc with a diameter of 7.8 µm, an edge thickness of 2.6 µm , and a central thickness of 0.8 µm. This shape maximizes the cell's surface area ( ~ 140 µm² ), an important attribute in gas exchange. Erythrocytes, or red blood cells, are the most abundant cell type in the blood. A microliter of blood contains about 5 million red blood cells, compared with only 4000–11,000 leukocytes and 150,000 450,000 platelets. In a healthy individual, approximately 1% of erythrocytes are removed from the circulation each day due to senescence (aging); however, bone marrow continuously produces new erythrocytes to replace those lost. The majority of aged erythrocytes (~90%) are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (~10%) break down intravascularly, releasing insignificant amounts of hemoglobin into the blood. The erythrocytes are extremely deformable. They pass easily through the narrowest capillaries by folding over on themselves.  Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide. Hemoglobin is the main component of red blood cells. Hemoglobin (Hb) is a large, complex protein with 4 globular protein chains. Each of which is wrapped around an iron-containing heme group. There are several isoforms of globin proteins in hemoglobin. The most common isoforms are designated alpha (a), beta (b), gamma (g), and delta (d), depending on the structure of the chain. Most adult hemoglobin (designated HbA) has two alpha chains and two beta chains. However, a small portion of adult hemoglobin (about 2.5%) has two alpha chains and two delta chains (HbA2). HEME SYNTHESIS & CATABOLISM Learning Objectives for Heme Synthesis and Catabolism ▪ Understand the Basics of Heme Structure and Function: Describe the chemical structure of heme and its role in biological systems, particularly in hemoglobin and cytochromes. ▪ Trace the Pathway of Heme Synthesis: Identify and explain the major steps in the biosynthesis of heme, including the key enzymes involved (e.g., ALA synthase, porphobilinogen deaminase). Discuss the regulation of heme synthesis and the impact of different physiological and pathological conditions. Recognize Heme Catabolism: Outline the process of heme catabolism, including the conversion of heme to biliverdin and subsequently to bilirubin HEME SYNTHESIS & CATABOLISM The four heme groups in a hemoglobin molecule are identical. Each consists of a carbon-hydrogen-nitrogen porphyrin ring with an iron atom (Fe) in the center. About 70% of the iron in the body is found in the heme groups of hemoglobin. Consequently, hemoglobin synthesis requires an adequate supply of iron in the diet. 1. Most dietary iron comes from red meat, beans, spinach, and iron-fortified bread. 2. Iron is absorbed in the small intestine by active transport. ❑ A carrier protein called transferrin binds iron and transports it in the blood(3). ❑ The bone marrow takes up iron and uses it to make the heme group of hemoglobin for developing red blood cells (4). Excess ingested iron is stored, mostly in the liver. Iron stores are found inside a spherical protein called molecule ferritin. The core of the sphere is an iron-containing mineral that can be converted to soluble iron and released when needed for hemoglobin synthesis. Excess iron in the body is toxic, and poisoning sometimes occurs in children when they ingest too many vitamin pills containing iron. Initial symptoms of iron toxicity are gastrointestinal pain, cramping, and internal bleeding, which occurs as iron corrodes the digestive epithelium. Subsequent problems include liver failure, which can be fatal. Red blood cells in the circulation live for about 120 days. Increasingly fragile older red blood cells may rupture as they try to squeeze through narrow capillaries, or they may be engulfed by scavenging macrophages as they pass through the spleen. Many components of the destroyed cells are recycled. Amino acids from the globin chains of hemoglobin are incorporated into new proteins, and some iron from the heme groups is reused to make new heme groups. The spleen and liver convert remnants of the heme groups to a colored pigment called bilirubin.  Bilirubin is carried by plasma albumin to the liver, where it is metabolized and incorporated into a secretion called bile. Bile is secreted into the digestive tract, and the bilirubin metabolites leave the body in the feces. Small amounts of other bilirubin metabolites are filtered from the blood in the kidneys, where they contribute to the yellow color of urine (7). In some circumstances, bilirubin levels in the blood become elevated (hyperbilirubinemia). This condition, known as jaundice, causes the skin and whites of the eyes to take on a yellow cast. The accumulation of bilirubin can occur from several different causes. Newborns whose fetal hemoglobin is being broken down and replaced with adult hemoglobin are particularly susceptible to bilirubin toxicity, so doctors monitor babies for jaundice in the first weeks of life. Another common cause of jaundice is liver disease, in which the liver is unable to process or excrete bilirubin. Normal heme biosynthesis — Heme is made in all tissues to serve as the prosthetic group for many essential hemoproteins. Heme synthesis is most active in the bone marrow and liver. Bone marrow – The bone marrow accounts for >80 percent of daily heme synthesis, primarily to provide heme for hemoglobin, the body's most abundant hemoprotein. Heme and globin synthesis are closely coordinated. The rate of heme synthesis is controlled by expression of the erythroid-specific gene (ALAS2), which encodes the first enzyme in the heme synthetic pathway, as well as genes for several other enzymes in the pathway. ALAS2 expression is upregulated by heme and by iron. Liver – The liver accounts for most of the rest of heme synthesis. Heme is used in the liver primarily in cytochrome P450 (CYP) enzymes, which metabolize toxins and drugs and turn over rapidly. Other tissues – Other important heme-containing proteins are present in all tissues. Examples include respiratory cytochromes, catalase, nitric oxide synthase, tryptophan pyrrolase, and myoglobin. Heme Iron redox state – Heme (iron protoporphyrin IX) is a complex molecule in which a single ferrous (Fe++) iron ion is surrounded by and coordinately bound to a protoporphyrin IX ring. If the iron is oxidized to the ferric state (Fe+++), the protein is called methemoglobin, and the affected molecule does not bind oxygen. Locations for heme synthesis – Heme synthesis occurs in all tissues but is especially prominent in bone marrow (used for synthesis of hemoglobin) and liver (used for hepatic cytochrome production). Synthesis occurs in the cytoplasm and mitochondria. HEME SYNTHESIS Synthesis of heme begins in mitochondria, with the formation of DELTA-AMINOLEVULINIC ACID (ALA) (an amino acid committed exclusively to the synthesis of heme) From GLYCINE and SUCCINYL-COA by ALA SYNTHASE (ALAS); ALAS is the first enzyme in the pathway that catalyzes a reaction between two simple molecules, glycine and succinyl-coenzyme A (succinyl-CoA), to form delta-aminolevulinic acid (ALA). ALAS requires pyridoxal-5'-phosphate (a derivative of vitamin B6 [pyridoxine]) as a cofactor. This step is rate-limiting in the liver. Hepatic ALAS is mainly regulated by heme via feedback repression (dashed arrow at the top of the frame).  ALAD (ALA dehydratase) – ALAD (also known as PBG [porphobilinogen] synthase) is the second enzyme in the heme synthetic pathway. In the cytoplasm, it catalyzes the synthesis of PBG, a pyrrole, from two molecules of ALA. ALA and PBG are commonly referred to as porphyrin precursors. They can accumulate in large amounts in the acute porphyrias and are associated with neurovisceral symptoms.  PBGD (porphobilinogen [PBG] deaminase, also known as hydroxymethylbilane synthase [HMBS]) – PBGD is the third enzyme in the pathway. It catalyzes the synthesis of hydroxymethylbilane (HMB; a linear tetrapyrrole) from four molecules of PBG. Pathogenic variants in the PBGD/HMBS gene cause Acute intermittent porphyria (AIP), which is symptomatic after puberty in some heterozygous individuals.  Uroporphyrinogen synthase (UROS) – UROS is the fourth enzyme in the pathway. It catalyzes the synthesis of uroporphyrinogen III (an octacarboxyl porphyrinogen) from HMB. The reaction results in cyclization of this linear tetrapyrrole and inversion of one pyrrole to form the first of the asymmetric porphyrins required for heme synthesis. Any remaining HMB cyclizes nonenzymatically to form uroporphyrinogen I, which is not a heme precursor. Pathogenic variants in the UROS gene cause Congenital erythropoietic porphyria (CEP), an autosomal recessive disorder characterized by accumulation of isomer I porphyrinogens, which undergo autooxidation to the corresponding isomer I porphyrins.  Uroporphyrinogen decarboxylase (UROD) – UROD is the fifth enzyme in the pathway. It catalyzes the 4-step decarboxylation of uroporphyrinogen III and I to form coproporphyrinogen III and I (tetracarboxyl porphyrinogens). Coproporphyrinogen oxidase (CPOX) – CPOX the sixth enzyme in the pathway. It is located in the mitochondria, where it catalyzes the synthesis of protoporphyrinogen IX from coproporphyrinogen III. Coproporphyrinogen I is not a substrate for this stereospecific enzyme, and as a result, only the isomer III porphyrinogen is metabolized to heme. Pathogenic variants in the CPOX gene cause hereditary coproporphyria (HCP) in some heterozygous individuals. Neurovisceral symptoms, and rarely blistering photosensitivity, can develop after puberty.  Protoporphyrinogen oxidase (PPOX) – PPOX is the seventh enzyme in the pathway. It oxidizes protoporphyrinogen IX by removing six protons to form protoporphyrin IX, which is the only oxidized porphyrin intermediate in the pathway. Ferrochelatase (FECH) – FECH is the eighth and final enzyme in the pathway. It inserts ferrous iron into protoporphyrin IX to form heme (iron protoporphyrin IX). Considerations important for diagnostic testing include: ALA and PBG – Elevations of ALA (delta-aminolevulinic acid) and PBG (porphobilinogen) are associated with neurovisceral manifestations in acute porphyrias. They are elevated in serum and urine but most concentrated in urine. A substantial PBG elevation is diagnostically specific for these acute porphyrias. Porphyrins -Urine/plasma – Urinary porphyrin elevations are seen during symptomatic acute porphyria episodes. Literatura e rekomanduar 1. Human Physiology Dee Unglaub Silverthorn 16.3 Red Blood Cells 16.3.1 Compare the structures of immature and mature red blood cells. 16.3.2 Describe the molecular structure of hemoglobin. 16.3.3 Create a map of iron metabolism and hemoglobin synthesis. 16.3.4 Describe the common pathologies of red blood cells. 2. UPTODATE 1. SUKSESE!