IU Indianapolis ScholarWorks :: Browsing by Author "Srour, Edward F."

Browsing by Author "Srour, Edward F."

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Absence of cardiomyocyte differentiation following transplantation of adult cardiac-resident Sca-1+ cells into infarcted mouse hearts
(American Heart Association, 2018-12-18) Soonpaa, Mark H.; Lafontant, Pascal J.; Reuter, Sean; Scherschel, John A.; Srour, Edward F.; Zaruba, Marc-Michael; Rubart-von der Lohe, Michael; Field, Loren J.; Medicine, School of Medicine
Although several lines of evidence suggest that the glycosyl phosphatidylinositol-anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT-EGFP; MHC-nLAC double-transgenic mice ([C57/Bl6J x DBA/2J]F1 genetic background; all procedures followed were in accordance with Institutional Guidelines). The ACT-EGFP transgene targets ubiquitous expression of an enhanced green fluorescent protein reporter, and the MHC-nLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, while cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-GAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme and the resulting cells reacted with an APC-conjugated anti-Sca-1 antibody and a PE-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage- cells were then isolated via fluorescence-activated cell sorting (FACS; characterization of the donor cells is provided in Figure 1A), and 100,000 cells were injected into the infarct border zone of non-transgenic [C57/Bl6J x DBA/2J]F1 mice immediately following permanent coronary artery occlusion.
Adult Bone Marrow–derived Cells Do Not Acquire Functional Attributes of Cardiomyocytes When Transplanted into Peri-infarct Myocardium
(Elsevier, 2008-06-01) Scherschel, John A.; Soonpaa, Mark H.; Srour, Edward F.; Field, Loren J.; Rubart, Michael; Microbiology and Immunology, School of Medicine
(BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca2+] i ) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lin– c-kit+ BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9–10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential–induced [Ca2+] i transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca2+] i transients were found to occur synchronously in host cardiomyocytes along the graft–host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart.
Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass
(Elsevier, 2019) Maupin, Kevin A.; Himes, Evan R.; Plett, Artur P.; Chua, Hui Lin; Singh, Pratibha; Ghosh, Joydeep; Mohamad, Safa F.; Abeysekera, Irushi; Fisher, Alexa; Sampson, Carol; Hong, Jung-Min; Childress, Paul; Alvarez, Marta; Srour, Edward F.; Bruzzaniti, Angela; Pelus, Louis M.; Orschell, Christie M.; Kacena, Melissa A.; Orthopaedic Surgery, School of Medicine
Osteoblast number and activity decreases with aging, contributing to the age-associated decline of bone mass, but the mechanisms underlying changes in osteoblast activity are not well understood. Here, we show that the age-associated bone loss critically depends on impairment of the ability of megakaryocytes (MKs) to support osteoblast proliferation. Co-culture of osteoblast precursors with young MKs is known to increase osteoblast proliferation and bone formation. However, co-culture of osteoblast precursors with aged MKs resulted in significantly fewer osteoblasts compared to co-culture with young MKs, and this was associated with the downregulation of transforming growth factor beta. In addition, the ability of MKs to increase bone mass was attenuated during aging as transplantation of GATA1low/low hematopoietic donor cells (which have elevated MKs/MK precursors) from young mice resulted in an increase in bone mass of recipient mice compared to transplantation of young wild-type donor cells, whereas transplantation of GATA1low/low donor cells from old mice failed to enhance bone mass in recipient mice compared to transplantation of old wild-type donor cells. These findings suggest that the preservation or restoration of the MK-mediated induction of osteoblast proliferation during aging may hold the potential to prevent age-associated bone loss and resulting fractures.
Ames hypopituitary dwarf mice demonstrate imbalanced myelopoiesis between bone marrow and spleen
(Elsevier, 2015-06) Capitano, Maegan L.; Chitteti, Brahmananda R.; Cooper, Scott; Srour, Edward F.; Bartke, Andrzej; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of Medicine
Ames hypopituitary dwarf mice are deficient in growth hormone, thyroid-stimulating hormone, and prolactin. The phenotype of these mice demonstrates irregularities in the immune system with skewing of the normal cytokine milieu towards a more anti-inflammatory environment. However, the hematopoietic stem and progenitor cell composition of the bone marrow (BM) and spleen in Ames dwarf mice has not been well characterized. We found that there was a significant decrease in overall cell count when comparing the BM and spleen of 4-5 month old dwarf mice to their littermate controls. Upon adjusting counts to differences in body weight between the dwarf and control mice, the number of granulocyte-macrophage progenitors, confirmed by immunophenotyping and colony-formation assay was increased in the BM. In contrast, the numbers of all myeloid progenitor populations in the spleen were greatly reduced, as confirmed by colony-formation assays. This suggests that there is a shift of myelopoiesis from the spleen to the BM of Ames dwarf mice; however, this shift does not appear to involve erythropoiesis. The reasons for this unusual shift in spleen to marrow hematopoiesis in Ames dwarf mice are yet to be determined but may relate to the decreased hormone levels in these mice.
C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis
(Wiley, 2016-04) Meijome, Tomas E.; Baughman, Jenna T.; Hooker, R. Adam; Cheng, Ying-Hua; Ciovacco, Wendy A.; Balamohan, Sanjeev M.; Srinivasan, Trishya L.; Chitteti, Brahmananda R.; Eleniste, Pierre P.; Horowitz, Mark C.; Srour, Edward F.; Bruzzaniti, Angela; Fuchs, Robyn K.; Kacena, Melissa A.; Orthopaedic Surgery, School of Medicine
C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.
CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche
(American Society of Hematology, 2014-07-24) Chitteti, Brahmananda Reddy; Kobayashi, Michihiro; Cheng, Yinghua; Zhang, Huajia; Poteat, Bradley A.; Broxmeyer, Hal E.; Pelus, Louis M.; Hanenberg, Helmut; Zollman, Amy; Kamocka, Malgorzata M.; Carlesso, Nadia; Cardoso, Angelo A.; Kacena, Melissa A.; Srour, Edward F.; Department of Medicine, IU School of Medicine
We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function.
Cell adhesion molecule CD166 drives malignant progression and osteolytic disease in multiple myeloma
(American Association for Cancer Research, 2016-12-01) Xu, Linlin; Mohammad, Khalid S.; Wu, Hao; Crean, Colin; Poteat, Bradley; Cheng, Yinghua; Cardoso, Angelo A.; Machal, Christophe; Hanenberg, Helmut; Abonour, Rafat; Kacena, Melissa A.; Chirgwin, John; Suvannasankha, Attaya; Srour, Edward F.; Microbiology and Immunology, School of Medicine
Multiple myeloma (MM) is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in MM cell lines and primary bone marrow (BM) cells from patients. CD166+ MM cells homed more efficiently than CD166− cells to the BM of engrafted immunodeficient NSG mice. CD166 silencing in MM cells enabled longer survival, a smaller tumor burden and less osteolytic lesions, as compared to mice bearing control cells. CD166 deficiency in MM cell lines or CD138+ BM cells from MM patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Further, CD166 deficiency in MM cells also reduced formation of osteolytic disease in vivo after intra-tibial engraftment. Mechanistic investigation revealed that CD166 expression in MM cells inhibited osteoblastogenesis of BM-derived osteoblast progenitors by suppressing RUNX2 gene expression. Conversely, CD166 expression in MM cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in MM cell homing to the BM and MM progression, rationalizing its further study as a candidate therapeutic target for MM treatment.
Characterization of Normal and Preleukemic Hematopoietic Stem Cell Responses to Physiologic and Extra-Physiologic Oxygen Tension
(2022-08) Aljoufi, Arafat; Kaplan, Mark H.; Zhang, Chi; Srour, Edward F.; Kapur, Reuben
Hematopoietic stem and progenitor cells (HSCs/HPCs) transplantation is a curative treatment for a variety of hematologic and non-hematologic diseases. Successful HSC transplantation requires infusing patients with a sufficient number of long-term engrafting HSCs. As a result, research efforts have focused on optimizing the collection process. Previous work established that harvesting mouse bone marrow HSCs under low oxygen tension similar to that reported for the bone marrow niche in situ (physioxia), results in enhanced HSC recovery and function. However, collecting bone marrow cells under physioxia is not a clinically viable approach. Here, I demonstrated that the collection and processing of peripheral blood mobilized with G-CSF alone or G-CSF and Plerixafor under physioxia resulted in a greater number of phenotypically defined long-term engrafting HSCs. Using high-resolution single cell sequencing to explore the molecular programs governing HSCs under physioxia, I identified increased expression of genes involved in HSC self-renewal and maintenance. In contrast, HSCs under ambient air upregulated genes implicated in HSC differentiation, apoptosis, and inflammatory pathways. Furthermore, wild-type HSCs under physioxia revealed a significant reduction in gene expression and activity of the epigenetic modifier Tet2. Consequently, I evaluated the phenotyping, engraftment potential and gene expression of preleukemic Tet2-/- bone marrow cells under physioxia and ambient air. Unlike wild-type HSCs, Tet2-/- HSCs/HPCs were unresponsive to changes in oxygen tension. Notably, we observed similar phenotypes, functions, and self-renewal and quiescence gene expression in wild-type HSCs under physioxia and Tet2- /- HSCs under physioxia or ambient air. These findings imply that the preserved stemness and enhanced engraftment of HSCs under physioxia may in part be a result of Tet2 downregulation. Understanding the mechanisms regulating wild-type and preleukemic HSCs under physioxia will have therapeutic implications for optimizing HSC transplantation and mitigating the growth advantage of preleukemic stem cells.
Clinical applications of thrombopoietin silencing: A possible therapeutic role in COVID-19?
(Elsevier, 2021-10) Alentado, Vincent J.; Moliterno, Alison R.; Srour, Edward F.; Kacena, Melissa A.; Medicine, School of Medicine
Thrombopoietin (TPO) is most recognized for its function as the primary regulator of megakaryocyte (MK) expansion and differentiation. MKs, in turn, are best known for their role in platelet production. Research indicates that MKs and platelets play an extensive role in the pathologic thrombosis at sites of high inflammation. TPO, therefore, is a key mediator of thromboinflammation. Silencing of TPO has been shown to decrease platelets levels and rates of pathologic thrombosis in patients with various inflammatory disorders (Barrett et al, 2020; Bunting et al, 1997; Desai et al, 2018; Kaser et al, 2001; Shirai et al, 2019). Given the high rates of thromboinflammmation in the novel coronavirus 2019 (COVID-19), as well as the well-documented aberrant MK activity in affected patients, TPO silencing offers a potential therapeutic modality in the treatment of COVID-19 and other pathologies associated with thromboinflammation. The current review explores the current clinical applications of TPO silencing and offers insight into a potential role in the treatment of COVID-19.
Defining the mechanism of prostaglandin E₂-enhanced hematopoietic stem and progenitor cell homing
(2014-04-02) Speth, Jennifer M.; Pelus, Louis M.; Broxmeyer, Hal E.; Harrington, Maureen A.; Ivan, Mircea; Srour, Edward F.
Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of hematological disorders. However, to be effective, transplanted HSCs must efficiently “home” to supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution, or in some cases, bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation efficiency. We have previously shown that ex vivo treatment of mouse or human HSCs with 16-16 dimethyl PGE2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting in part from upregulation of surface CXCR4 expression. We now show that pulse-treatment of mouse or human HSPCs with dmPGE2 stabilizes HIF1α in HSPCs, and that similar treatment with the hypoxia mimetic DMOG produces analogous effects to dmPGE2 on HSPC CXCR4 expression and homing. This suggests that HIF1α is responsible for PGE2’s enhancing effects on HSPCs. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP), confirms the requirement of HIF1α for dmPGE2-enhanced migration and CXCR4 upregulation. Additionally, we confirm the requirement for HIF1α in dmPGE2-enhanced in vivo homing using a conditional knockout mouse model of HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after PGE2 treatment. Interestingly, we also observe an increase in the small GTPase Rac1 after dmPGE2 treatment, as well as a defect in PGE2-enhanced migration and CXCR4 expression in Rac1 knockout HSPCs. Using state-of-the-art imaging technology we, confirm an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment that likely explains enhanced sensitivity of PGE2-treated HSPCs to SDF-1. Taken together, these results define a precise mechanism through which ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing, and describe a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

Browsing by Author "Srour, Edward F."

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