Increased expression of HIF2α during iron deficiency–associated megakaryocytic differentiation

Summary Background Iron deficiency is associated with reactive thrombocytosis; however, the mechanisms driving this phenomenon remain unclear. We previously demonstrated that this occurs alongside enhanced megakaryopoiesis in iron‐deficient rats, without alterations in the megakaryopoietic growth factors thrombopoietin, interleukin‐6, or interleukin‐11. Objectives The aim of this study was to evaluate megakaryocyte differentiation under iron deficiency in an in vitro model and to investigate potential genes involved in this process. Methods Human erythroleukemia and megakaryoblastic leukemia cell lines, as well as cord‐blood derived hematopoietic stem cells were cultured under iron deficiency. Cell morphology, ploidy, expression of CD41, CD61, and CD42b, and proplatelet formation were assessed in iron‐deficient cultures. Polymerase chain reaction arrays were used to identify candidate genes that were verified using real‐time polymerase chain reaction. Hypoxia‐inducible factor 1, α subunit (HIF2α) protein expression was assessed in bone marrow sections from iron‐deficient rats and vascular endothelial growth factor (VEGF)‐A in culture supernatants. Results and Conclusions Iron deficiency enhanced megakaryoid features in cell lines, increasing ploidy and initiating formation of proplatelet‐like structures. In cord blood cell cultures, iron deficiency increased the percentage of cells expressing megakaryopoietic markers and enhanced proplatelet formation. HIF2α and VEGF were identified as potential pathways involved in this process. HIF2α protein expression was increased in megakaryocytes from iron‐deficient rats, and VEGF‐A concentration was higher in iron‐deficient culture supernatants. Addition of VEGF‐A to cell cultures increased percentage expression of megakaryocyte CD41. In conclusion, the data demonstrate that iron deficiency augments megakaryocytic differentiation and proplatelet formation and a potential role of HIF2α in megakaryopoiesis.

Summary. Background: Iron deficiency is associated with reactive thrombocytosis; however, the mechanisms driving this phenomenon remain unclear. We previously demonstrated that this occurs alongside enhanced megakaryopoiesis in iron-deficient rats, without alterations in the megakaryopoietic growth factors thrombopoietin, interleukin-6, or interleukin-11. Objectives: The aim of this study was to evaluate megakaryocyte differentiation under iron deficiency in an in vitro model and to investigate potential genes involved in this process. Methods: Human erythroleukemia and megakaryoblastic leukemia cell lines, as well as cord-blood derived hematopoietic stem cells were cultured under iron deficiency. Cell morphology, ploidy, expression of CD41, CD61, and CD42b, and proplatelet formation were assessed in iron-deficient cultures. Polymerase chain reaction arrays were used to identify candidate genes that were verified using real-time polymerase chain reaction. Hypoxia-inducible factor 1, a subunit (HIF2a) protein expression was assessed in bone marrow sections from iron-deficient rats and vascular endothelial growth factor (VEGF)-A in culture supernatants. Results and Conclusions: Iron deficiency enhanced megakaryoid features in cell lines, increasing ploidy and initiating formation of proplatelet-like structures. In cord blood cell cultures, iron deficiency increased the percentage of cells expressing megakaryopoietic markers and enhanced proplatelet formation. HIF2a and VEGF were identified as potential pathways involved in this process.

Introduction
Platelets play a fundamental role in hemostasis and thrombosis and an emerging role in inflammation and cancer biology. Platelet production occurs at a rate of 10 11 platelets daily and may increase up to 20-fold in response to high demand [1]. Reactive thrombocytosis occurs in response to infection, tissue damage as occurs during surgery, chronic inflammation, malignancy, and post splenectomy [2] and is by far more common than primary thrombocytosis. Another well-known cause of reactive thrombocytosis is iron deficiency (ID) [3][4][5][6]. While the elevation of platelet count is usually mild to moderate in ID (~500 9 10 9 L À1 ), platelet counts reaching 1000 9 10 9 L À1 have been reported [3,4].
The mechanism behind this phenomenon is not fully elucidated; however, animal models of ID-including a diet-induced ID model established by our group-show that ID alone leads to increased platelet count [7][8][9]. This is accompanied by changes in megakaryopoiesis, such as enhanced progenitor expansion, increased megakaryocyte (MEG) ploidy, and overall augmented MEG differentiation. Furthermore, platelets produced in ID had higher aggregability [9], suggesting that elevated platelet count in ID may not be entirely risk free. There are numerous case reports linking ID-related thrombocytosis and thrombosis [10] and several case-control studies showing that ID is more common in patients with a cerebrovascular insult in comparison to controls [11][12][13].
Anemia is a common complication in inflammatory bowel disease (IBD) (6.5-73.7%) [14] and in cancer (30-90%) [15]; in both cases, ID appears to be the predominant cause [14][15][16][17]. Thrombocytosis is not uncommon in either condition [18,19], and both cancer and IBD patients have increased risk for venous thromboembolism (VTE) [20][21][22][23], for which, in the case of cancer, high platelet count is an independent risk factor [20,21]. Iron replacement therapy in patients with IBD and ID anemia normalizes platelet count and reduces platelet activity [24,25]. While it is unclear if this affects risk for thromboembolic events in the long term, anemic cancer patients have a diminished incidence of VTE if erythropoiesis-stimulating agents are administered with intravenous iron [26] as opposed to no-iron therapy. This suggests that ID-related thrombocytosis may be of relevance in the clinical setting as well.
Megakaryocytes arise from hematopoietic stem cells, and share a common progenitor with erythrocytes [27]. Megakaryocyte differentiation is marked by augmented expression of CD41, CD61, and CD42b, while erythroid markers such as glycophorin A are downregulated [27]. Megakaryocytes undergo endomitosis, initiating multiple cycles of DNA replication without completing cytokinesis [28], allowing ploidy to increase geometrically from 2n to 64n. This functional gene amplification facilitates the ensuing increase in protein and lipid synthesis and substantial cell enlargement required for platelet biogenesis and function [29]. An MEG may produce platelets at any ploidy level, although higher ploidy level may correlate with higher platelet production [30]. Megakaryocyte terminal differentiation involves migration from the hypoxic osteoblastic niche toward bone marrow sinusoids [31], where cytoplasmic projections termed proplatelets are extended into the bloodstream and then fragment into platelets (reviewed in [32]).
Iron is critical for cell survival, growth, and differentiation and is a functional component of heme-and ironsulfur-cluster proteins involved in mitochondrial function, catalysis, redox reactions, DNA replication, and transcription (reviewed in [33]). There is not much known about iron homeostasis in MEGs and how ID affects megakaryopoiesis. Cytokines more commonly associated with mediating megakaryopoiesis such as thrombopoietin, interleukin (IL)-6, and IL-11 are not altered in patients with ID anemia and thrombocytosis [24,34] or in our animal model of ID [9].
In this study, we investigated potential targets that may be involved in ID megakaryopoiesis in an in vitro model of ID. ID enhanced MEG features in cell lines and in cord blood-derived hematopoietic stem cells (CBHSCs) and modulated the genes involved in the hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) pathways.

CBHSC isolation and culture
Cord blood was obtained from the Department of Obstetrics and Feto-Maternal Medicine after approval by the Ethics Commission of the Medical University of Vienna. CD34 + CBHSCs were isolated by using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), cultured for 6-7 days in serum-free medium (X-vivo 15; Lonza, Szabo, Vienna, Austria) with 50 ng mL À1 thrombopoietin (TPO; Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan), and transferred to IR (100%) or ID (1.25%) serum-free medium with 100 ng mL À1 TPO for 5 days.
Megakaryocyte differentiation was assessed by flow cytometric measurement of CD41, CD61, and CD42b surface expression (see Data S1). The percentage of positive staining cells was determined using flow cytometry, and the median fluorescence intensity (MFI) was based on the gated positive cell population of each MEG marker. Ploidy was measured as in cell lines, with anti-CD41 to gate for MEGs. VEGF-A in cell supernatants was assessed via immunoassay (ProcartaPlex, eBioscience, Vienna, Austria) measured on a BioPlex 200 (BioRad; Hercules, CA, USA). Cell counts after culture in IR and ID media supplemented with 25 ng mL À1 VEGF (eBioscience), 1 ng mL À1 erythropoietin (EPO; eBioscience), or 100 ng mL À1 TPO were performed after staining with anti-CD41-FITC (MEG) and anti-glycophorin A-PE-Cy5 (erythrocyte) conjugated antibodies (eBioscience). At least 100 cells per condition were counted under 940 magnification. To calculate percentages, the number of green (CD41) or red (glycophorin A) cells were divided by the total number of visible nuclei (see Data S1).

Proplatelet formation
Proplatelet formation and assessment at day 5 were based on previously described methods [35,36] (see Data S1). Fixed samples were incubated with mouse anti-a-tubulin (Sigma-Aldrich) followed by anti-mouse-IgG conjugated to Alexa488 (Invitrogen/Life Technologies), and DAPI for nuclear counterstaining. Images (910) were taken with an Axioimager M2 and analyzed using Zen lite software (Zeiss, Munich, Germany; Fig. S1).

Real-time polymerase chain reaction analysis of gene expression
Real-time polymerase chain reaction (RT-PCR) was performed using a TaqMan array with customized targets and a catalogued array for transcription factors (Applied Biosystems/Life Technologies, Lofer, Austria). RNA was pooled from two independent experiments after 1 day (HEL) or 2 days (CMK) of culture in ID (slower growth of CMK compared with HEL). Three independent 5-day experiments of CD61 + MACS sorted MEGs were pooled. HPRT1 and 18S were used as endogenous controls, and cut-offs were set at 2.0 for upregulation and 0.5 for downregulation. Individual genes such as TfR1, HIF2a, HIF1a, VEGFA (Qiagen, Hilden, Germany), VEGFR1, and VEG-FR2 (VBC Biotech, Vienna, Austria) were verified using SYBR green (Applied Biosystems/Life Technologies). Analysis was performed using Applied Biosystems software and LinRegPCR [37]. Relative expression was calculated using 2 ÀðCT Target À ; CT Housekeeping Þ and relative quantity using 2 ÀðDCT ID ÀDCT IR Þ .
Immunohistochemical staining DAB-HRP staining was performed on paraffin-embedded sections of rat sterna from the rat model of ID [9] after incubation with anti-HIF2a (Novus Biologicals, Abingdon, UK). Images of slides with covered labels were randomized and evaluated in ImageJ. Individual MEGs were evaluated based on staining intensity from 0 (no staining) to 3+ (intense staining). Immunohistochemical score was calculated by summing the product of percentage total MEGs and score number. Blood was drawn from the sublingual vein and analyzed on a Cell-Dyn 3500 analyzer (Abbott Diagnostics, Abbot Park, IL, USA).

Statistical testing
Statistical testing was performed using t-test for independent samples, one-sample t-test with a test value of 1 for fold changes, ANOVA with Tukey post-hoc testing, and the Mann-Whitney U Test in SPSS 21.

Results
Iron deficiency induces megakaryocytic differentiation in HEL and CMK HEL and CMK cell lines were selected because both are capable of megakaryocytic differentiation on induction by PMA [38][39][40]. HEL is derived from a patient with erythroleukemia [41] and is less mature in comparison to CMK, which is derived from a patient with megakaryoblastic leukemia [42], thus allowing insight into different stages of megakaryopoiesis.
Culture in media without iron led to diminished proliferation in both HEL and CMK in comparison to fully IR media (Fig. 1A), and area under the curve (AUC) analysis confirmed this difference (Fig. S2A). Proliferation was arrested at day 11 and thus excluded from analysis. The 1.25% condition was selected for ID as it allows for cell proliferation, while still rendering cultures iron deficient. TfR1, which is stabilized when iron is low [43], is upregulated in ID in both cell lines on RT-PCR ( Fig. 1B), beginning after 1 day of culture and reaching a maximum after 2 days in HEL and 7 in CMK. Flow cytometric measurement showed an increased surface TfR1 expression in HEL and CMK; however, the differences were not as distinct as on RT-PCR (Fig. S2B).
Culture in ID resulted in distinct morphological changes similar to those observed on megakaryocytic differentiation by PMA (Fig. 1D). Cell volume was increased in HEL after 4 days of culture in ID (Fig. 1D, arrowheads; Fig. 2C, P = 0.008) and IR plus 50 nmol L -1 PMA (P < 0.001) compared with IR alone (Fig. 1D, arrowheads). In CMK, volume was increased in PMA treatment (P = 0.001) but not in ID (P = 0.95).
The percentage of HEL cells with a ploidy greater than 4n increased almost 4-fold on flow cytometry (Fig. 2B, D). This matches the larger nuclei seen in fluorescence microscopy ( Fig. 2A). Similar changes occurred in HEL treated with 50 nmol L -1 PMA. CMK, in contrast, did not increase in ploidy under ID and showed only a trend to increased ploidy on PMA treatment and no apparent differences in nuclear size. These changes in ploidy in ID corroborate previous data [9]. In addition, proplatelet-like cytoplasmic extensions formed in CMK under ID (P = 0.01, Fig. 1C, D, arrows), which were similar to the changes observed on treatment with 50 nmol L -1 PMA (P < 0.001). Addition of 50 nmol L -1 PMA to ID media did not show additive effects in ploidy or cell volume in HEL (Fig. S3C) and was lethal in CMK, preventing analysis (Fig. S3A). Thus, ID may influence different aspects of megakaryopoiesis, augmenting endomitosis and later events such as proplatelet formation.

Iron deficiency potentiates MEG differentiation in CBHSCs
To investigate megakaryopoiesis under ID in primary cells, CBHSCs were cultured in IR or ID medium. Induction of ID was confirmed by increased TfR1 expression on RT-PCR for TfR1 (Fig. 3A).
ID increased the total number of proplatelets formed, the total number of cells that adhered to the fibrinogencoated slide, and the number of cells actively forming proplatelets (Fig. 3D). The ratio of proplatelet-forming MEGs to total MEGs had a trend toward increase under ID (Fig. S4D). ID did not appear to increase the number of proplatelets formed per MEG (Fig. S4D).

Iron deficiency differentially regulates gene expression in HEL, CMK, and MEGs
Custom array targets were selected to include genes involved in iron homeostasis as well as hematopoiesis, megakaryopoiesis, and erythropoiesis. Cell cycle regulators were also included, because ID may influence endomitosis. We included genes involved in apoptosis, because ID influences apoptosis and platelet production is influenced by apoptotic pathways. Inclusion of genes involved in the generation of reactive species and antioxidant enzymes was based on the role of iron in oxidative stress (reviewed in [44]). We were interested in the HIF pathway regulating response to hypoxic stress, which occurs during ID anemia and is potentially regulated by iron. HIF2a, in particular, is involved in the regulation of erythropoiesis under hypoxia and regulates targets involved in iron homeostasis (reviewed in [45]). We also used a second preassembled array for transcription factors (Table S1).
Of 187 genes from the RT-PCR arrays, only 23 were found to be regulated in HEL, none in CMK, and three in MEGs (Fig. 4, Table S1). TfR1 was increased in all three cell types in ID; however, only HEL reached the set cut-off, which indicates this cut-off is stringent (Fig. 4B). Of the regulated targets, only HIF2a was commonly upregulated in HEL and MEGs above 2-fold. VEGF-R1 was increased in both HEL and MEGs; however, the latter only had a 1.75-fold change. VEGF-R2 was upregulated in MEGs but downregulated in HEL.
Apart from TfR1, several genes involved in iron homeostasis were regulated by ID. Heme oxygenase 1, which is inducible and previously identified as strongly responsive to ID and oxidative stress [46], was downregulated in HEL but upregulated in MEG (Fig. 4B). Heavy chain ferritin (FTH) and SMAD1, a regulator of hepcidin, a key player in iron homeostasis [33], were also upregulated in HEL.
In HEL, transcription factors involved in hematopoietic and megakaryocytic differentiation such as TGIF1 [47], ELF-1 [48,49], and GATA-6 [48], were upregulated, while FOS was downregulated. Treatment of HEL cells with testosterone increases the expression of thromboxane A2 receptor [50], suggesting that the ID-induced increase in androgen receptor may have consequences for platelet function (Fig. 4B). Antiapoptotic gene BCL2 may influence cell survival and transition from endomitosis to platelet production. The specific roles of ATF3, FOXO1, FOXA2, and SMAD9 in the context of ID-megakaryopoiesis are unclear.
We were interested in the regulation of HIF2a, VEG-FR1, and VEGFR2 by ID because HIF2a coordinates oxygen and iron status with erythropoiesis and the VEGF pathway has been shown to influence various aspects of megakaryopoiesis [51][52][53]. Of the identified targets, only these were similarly regulated in HEL and MEGs. Additionally, VEGF is under HIF regulation. To verify the expression of genes in HIF/VEGF pathway in ID, HEL and CMK were cultured under ID for 3 days and CBHSCs for 5 days prior to RT-PCR (Fig. 5). TfR1 was upregulated in ID in all cell types. HIF2a was also upregulated in all cell types. VEGFR1 was upregulated in HEL, and CMK but not in MEGs and CD61-negative cells. VEGFR2 was upregulated in HEL, CMK, and MEGs but not in CD61-negative cells. VEGFA, a known downstream target of the HIF pathway and ligand to these receptors [54,55], was increased in all cell types, despite not being identified in the previous screen. HIF1a was upregulated in HEL, CMK, and CD61-negative cells but not in MEGs. Overall, the data suggest a role for HIF2a and VEGFA in megakaryocytic differentiation in ID.
Because previous studies have shown that VEGF-A influences megakaryopoiesis [51][52][53] and because VEGF-A is a known target of HIF2a [54,55], we evaluated the concentration of VEGF-A in the cell media of MEGs. The concentration of VEGF-A in media from ID cultures increased almost 2-fold (Fig. 6A). When cells were cultured (48 hours) in IR and ID media supplemented with VEGF-A (25 ng mL À1 ), the percentage of CD41+ cells per field of view increased in VEGF-treated cultures in comparison to TPO-treated cultures, but not the percentage of GPA+ cells (Fig. 6B, C). EPO (1 ng mL À1 )-supplemented cultures had a higher concentration of GPA+ cells in IR compared with ID (P = 0.004); conversely, CD41 + cell concentration was higher in ID (P < 0.001). While we did not see changes between TPO-treated ID and IR cultures at this early timepoint, CD41 + cell concentration was increased after 5 days of culture (P = 0.029, Fig. 6B, Fig. S5). EPO and VEGF-A cultures did not survive up to day 5, preventing analysis at this timepoint. These results support ID-driven megakaryopoiesis, where EPO exacerbates ID leading to a higher percentage of CD41 + cells. Furthermore, the increase in percentage CD41 + with VEGF in comparison to TPO and the lack of difference between ID and IR suggest that VEGF may have a role in this process.

HIF2a expression is increased in iron deficiency and correlates with platelet count
To determine if protein levels of HIF2a were increased in vivo as well, we used sternal sections of ID and control rats from our previous experiments [9] for IHC analysis. In this animal model, maintenance on a diet low in iron over the course of 3 weeks resulted in increased platelet counts (P < 0.001), as well as diminished hemoglobin (Hb, P < 0.001) and mean corpuscular volume (MCV, P < 0.001) (Fig. 7A), which corresponds with the microcytic hypochromic anemia induced by ID in humans. Erythrocyte count and hematocrit were likewise diminished in ID (Fig. S6A), and leukocyte count was unchanged (Fig. S6A). HIF2a was present in MEGs (Fig. 7B, red arrows) and non-MEGs in control rats, while staining intensity was diminished in non-MEGs in most sections and increased in MEGs in ID rats (Fig. 7B, green arrows, see Fig. S6B for isotype control). IHC scoring confirmed increase in staining intensity upon ID (P = 0.008) as compared with control rats (Fig. 7B, C). Plots of platelet counts against IHC score show two separate clusters corresponding to control and ID animals (Fig. 7C) and indicate increased HIF2a with higher platelet counts in ID.

Discussion
In this study, we utilized various in vitro models to examine the effect of ID on megakaryopoiesis and to identify potential targets involved in this process. We show that ID induces megakaryocytic differentiation and proplatelet formation. The novel finding of this study is identification of the HIF/VEGF pathways as potential regulators of megakaryocytic differentiation under ID. Apart from TfR1, the expression of genes from the HIF and VEGF pathways were identified by RT-PCR arrays to be induced upon ID. The induction of HIF2a was verified in bone marrow of ID animals as was the secretion of VEGF-A in supernatants of CBHSC. VEGF-A supplementation increased MEG percentages regardless of iron concentration, suggesting involvement in iron deficient megakaryopoiesis.
Our lab has previously demonstrated increased ploidy in iron deficient megakaryocytic cell lines [9]. Here, we confirmed ploidy increase in HEL, corroborated by increased expression of cyclin D, a cell cycle regulator whose overexpression increases MEG ploidy [56]. Early stages of megakaryopoiesis are associated with an increase in cell cycle regulators, and initial low expression of genes involved in MEG terminal differentiation [57,58]. This pattern of expression correlates with the pattern seen in HEL, where ID induces ploidy, while genes involved in terminal differentiation are diminished. There was also upregulation of ELF-1, a transcription factor progressively upregulated in megakaryopoiesis and downregulated in erythroid terminal differentiation [48,49], and downregulation in beta hemoglobin, supporting the shift away from erythropoiesis. Further transcription factors involved in differentiation were also regulated, however only in HEL.
Rats on ID-diet develop thrombocytosis, with bone marrow alterations suggesting not only increased MEG ploidy, but also MEG progenitor expansion, and enhanced differentiation [9]. This matches our observed expansion of the MEG population expressing early and late markers of megakaryopoiesis, and enhanced proplatelet formation in both CBHSC and CMK. Furthermore, the proportion of MEGs readily producing proplatelets is increased in ID. Ploidy was not increased in CBHSCs, but it is known that cord blood-derived MEGs do not become highly polyploid [30] and ID-enhanced endomitosis may still occur in bone marrow. CMK is a more mature cell line than HEL [41,42] and, hence, exhibited induction of later features of megakaryopoiesis instead of ploidy. These changes are accompanied by the expression of CD61 and vWF, which increase in terminal differentiation [58]. While we did not see regulation of these genes in MEGs in these genes, this is likely a limitation of prior sorting for CD61 expression.
Previous studies suggest that TPO, IL-6, and IL-11 may not be the primary mediators of ID-augmented megakaryopoiesis [9,24]. We identified VEGF as a potential pathway involved, as VEGFR2 and VEGFA were modulated by ID and immunoassay of cell media showed increased VEGFA concentrations in ID. Addition of VEGF to culture media also increases the percentage of cells expressing CD41, with no additive effect of ID. Stimulation of VEGFR2 induces the expression of megakaryocytic markers [53], while VEGFR1 increases MEG ploidy [51,52]. Megakaryoid and erythroid precursors both secrete VEGF, which may then stimulate MEG differentiation in an autocrine and paracrine fashion [51,53]. Thus, ID could increase both receptor and ligand expression, leading to augmented MEG polyploidization and differentiation. Furthermore, stimulation of this pathway has been shown to increase the number of circulating platelets in vivo, by way of increased MEG migration toward sinusoids in the bone marrow [52].
HIFs are central regulators of the response to hypoxic stress. These transcription factors are regulated by the availability of iron and oxygen, two factors diminished in ID anemia. Furthermore, HIFs are known regulators of the VEGF pathway [54,55], and HIF2a is also known to regulate cyclin D [59]. Another important HIF2a target is EPO, allowing regulation of erythropoiesis and iron absorption in response to changes in oxygen and iron [60]. We hypothesize that alterations in this pathway promote megakaryopoiesis. As MEGs retain HIF2a expression in ID, this could allow sustained expression of VEGFA, which facilitates megakaryopoiesis and MEG migration toward sinusoids, while erythropoiesis is stalled due to lack of iron for hemoglobin.
This study warrants further investigations into the HIF/VEGF pathways under ID. Our in vitro model does not take into account the bone microenvironment, which is involved in the regulation of megakaryopoiesis. Furthermore, ID in vivo progressively influences oxygenation and, while it is striking that ID alone can invoke such changes, the situation in vivo is likely more complex. In  , glycophorin A (red, arrows), and DAPI nuclear staining after culture in IR and ID media supplemented with 100 ng mL À1 TPO (control), 25 ng mL À1 VEGF, and 1 ng mL À1 EPO for 2 days. The results from two to five independent experiments are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
fact, hypoxia alone modulates megakaryopoiesis in rats, leading to increases in MEG volume, number, platelet count, and platelet aggregability [61]. Thus, ID and hypoxia may work in concert to augment megakaryopoiesis. ID may also influence intracellular redox balance, and megakaryopoiesis as well as hematopoiesis in general are greatly influenced by reactive oxygen species production (see [62,63] for reviews).
In conclusion, ID has a direct effect on megakaryopoiesis, augmenting MEG ploidy, as well as overall differentiation and subsequent proplatelet production. ID modulates gene expression, leading to increased HIF2a (B) Representative images of bone marrow IHC for HIF2a from control and iron-deficient rats. Red arrows depict megakaryocytes in control mice with less-intense HIF2a staining, and green arrows depict megakaryocytes in ID mice with more-intense staining. (C) Percentage of total control (n = 1735) and iron-deficient (n = 1621) bone marrow megakaryocytes under each staining intensity and the median calculated IHC score for control (n = 5) and iron-deficient (n = 5) rats. (D) Scatterplots of platelet count against IHC score. ***P ≤ 0.001. FOV indicates field of view. and increased expression of VEGFA. This would, in theory, lead to increased platelet production that would facilitate coagulation in the context of chronic bleeding. However, this could also increase the risk of thromboembolic events in patients suffering from chronic ID, particularly in IBD or cancer. Further investigation into megakaryopoiesis in ID is necessary.

Acknowledgements
The financial support by the Federal Ministry of Economy, Family and Youth, and the National Foundation of Research, Technology and Development is gratefully acknowledged. This project was funded by the Austrian Science Fund (project 21200). We are grateful to the Medical University of Vienna Core Facility, genomics and imaging units, for the analysis of RNA quality and the use of the Axioimager. We also acknowledge the Department of Obstetrics and Feto-Maternal Medicine for their cooperation. Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan is acknowledged for contributing human recombinant thrombopoietin.

Disclosure of Conflict of Interest
C. Gasche reports grants from AOP Pharmaceuticals and Biogena, as well as personal fees and nonfinancial support from Vifor International, outside the submitted work. All other authors state that they have no conflict of interest.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Fig. S1. Proplatelet quantification. Fig. S2. Iron deficiency increases TfR1 expression in HEL and CMK. Fig. S3. Iron deficiency does not augment the effect of PMA. Fig. S4. Iron deficiency increases the population of cells expressing megakaryocyte markers without altering ploidy.  and iron-deficient (n = 8) rats after 3 weeks. Table S1. DDC t of all investigated targets on qPCR arrays Data S1. Supplementary methods.