Cytomegalovirus Results in Poor Graft Function via Bone Marrow-Derived Endothelial Progenitor Cells

doi:10.21203/rs.3.rs-4353689/v1

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Cytomegalovirus Results in Poor Graft Function via Bone Marrow-Derived Endothelial Progenitor Cells

https://doi.org/10.21203/rs.3.rs-4353689/v1

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Poor graft function (PGF), characterized by myelosuppression, represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However, the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) can support hematopoiesis. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia (HCMV-emia) accompanied by PGF as well as after infected by HCMV AD 169 strain in vitro. We found dysfunction of HCMV-infected EPCs was characterized by decreased cell proliferation, tube formation, migration and hematopoietic support, and increased apoptosis and secretion of TGF-β1. We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover, HCMV up-regulates the p38 MAPK and its downstream transcription factor AP-1 to induce myelosuppression through promoting TGF-β1 secretion. In conclusion, HCMV could infect BM-EPCs and lead to their dysfunction. Enhanced secretion of TGF-β1 by BM-PECs is induced by HCMV through the up-regulation of p38 MAPK and its downstream transcription factor AP-1, resulting in myelosuppression, which might make a substantial contribution to the pathogenesis of PGF after allo-HSCT.

human cytomegalovirus

endothelial progenitor cells

poor graft function

transforming growth factor beta

allogeneic hematopoietic stem cell transplantation

Allogeneic stem cell transplantation (allo-HSCT) is a well-established therapeutic modality aimed at achieving a cure for individuals diagnosed with hematological malignancies or non-malignant disorders [1, 2]. Poor graft function (PGF) constitutes a significant adverse outcome subsequent to allo-HSCT with poor prognosis characterized by severe cell depletion in at least two lines of erythroid, granuloid and megakaryocyte [1, 2]. Nevertheless, the etiology of PGF remains obscure and the therapeutic effect is not satisfactory.

Relevant clinical studies have proved that cytomegalovirus (CMV) plays a significant role in the occurrence PGF [4, 5] and results in abnormity of hematopoietic reconstitution post-transplant. [6, 7] CMV can cause hemopoietic inhibition by infecting bone marrow stem progenitor cells and bone marrow stromal cells [8–11]. Bone marrow-derived endothelial progenitor cells (BM-EPCs) play a pivotal role as essential cellular components within the stromal cells residing in the bone marrow (BM) microenvironment, exerting a significant influence on supporting hematopoiesis [9, 12]. New findings from studies conducted on mice and humans suggest that the abnormality of EPCs is linked to development of PGF [13, 14]. Furthermore, BM-EPCs possess the capacity to secrete an array of hematopoietic-related cytokines [15, 16]. Nevertheless, there is currently limited knowledge regarding the impact of HCMV on BM-EPCs, particularly on their ability to support hematopoiesis.

Mitogen-activated protein kinases (MAPKs), a group of enzymes belonging to the serine/threonine kinase family, comprises three primary subgroups: extracellular signal-regulated kinase (ERK), p38 MAPK (p38), and JNK [2]. The impaired functionality in cultured EPCs in PGF patients has been reported to be significantly regulated by the crucial involvement of p38 MAPK [2], which indicates the significant involvement of MAPKs in development of PGF. One of the most important MAPK substrates is the transcriptional factor activator protein−1 (AP−1) [17 ], a transcription factor comprising dimers formed by Jun and Fos families [18, 19], which could be regulated by human CMV (HCMV) [20]. However, the participation of the MAPK pathway and/or AP−1 in the regulation of the hematopoietic related dysfunction of BM-EPCs after HCMV infection remains unknown.

We propose that the pathogenesis of HCMV-induced PGF involves modulation of BM-EPCs. To verify this hypothesis, our study investigated the impact of HCMV on BM-EPCs and elucidated the underlying mechanism.

Viruses

The generous contribution of Human Embryo Fibroblasts (HF) and Recombinant HCMV Strain AD169 was provided by Professor Guojun Wu (Xiangya Medical College of Central South University). A single stock of AD169 virus at 3 x 10⁷ plaque-forming units (PFUs)/ml was used throughout the subsequent experiment.

Isolation, cultivation and identification of BM-EPCs

Isolation, cultivation and identification of BM-EPCs were according to literature [2]. Briefly, the BM mononuclear cells (MNCs) were separated using density gradient centrifugation, a technique employed for cell separation. Subsequently, they were cultured with EGM-2-MV-SingleQuots from Lonza in Walkersville, MD for a duration of 7 days prior to undergoing testing. After one week of cultivation, the BM adherent cells were identified as EPCs through the utilization of Flow cytometry to detect CD34, CD133, and vascular endothelial growth factor receptor 2 (CD309). Dil-Acetylated Low Density Lipoprotein (DiI-AcLDL) uptake and fluorescein isothiocyanate–labeled Ulex Europaeus Agglutinin-I (FITC-UEA-I) binding assay were also used to identify the characterization of EPCs and assess the quantities of EPCs exhibiting dual positive staining as previously described [2]. At day 7 of cultivation, EPCs were characterized by the expression of CD34, CD309, and CD133 and their capacity to uptake DiI-AcLDL and bind FITC-UEA-I.

HCMV infects EPCs and HCMV detection

Isolation, cultivation and identification of BM-EPCs were according to literature [2]. For infection in vitro, the adherent EPCs from healthy donors of cultivation at 1–2 passage were exposed to HCMV AD169 at a multiplicity of infection (MOI) of 5 PFUs per cell for a duration of two hours. Following infection, phosphate-buffered saline (PBS) was employeed to rinse cells in order to eliminate any remaining unabsorbed virus. Continuous cultures were established for 96 hours, and EPCs were collected for the following experiments. EPCs from health donors which were not infected by HCMV were used as normal controls.

Indirect immunofluorescence (IF) was performed to detect HCMV pp65 in EPCs. HCMV-DNA of plasma in human subjects was quantified using quantitative real-time polymerase chain reaction (QT-PCR), and the threshold for HCMV-DNA was < 500 copies/ml [21].

Assessment of cell proliferation, apoptosis and angiogenesis of BM-EPCs

The adherent EPCs in culture were rinsed using PBS and gently released using trypsin supplemented with 0.125% EDTA. The analysis of BM-EPCs encompassed assessment of cellular proliferation, evaluation of migratory capacity, examination of tube formation potential, and investigation of apoptosis, was performed in accrodance to the previous investigation [2].

Coculture of BM CD34 + cells with EPCs

A CD34 MicroBead Kit was used to isolated BM CD34⁺ HSCs from healthy donors. The isolated CD34 + cells were cultured in StemSpan SFEM without direct contact and separated from adherent EPCs using 0.4-mm transwell inserts for a duration of 4 days. The hematopoietic ability of BM CD34 + cells was determined by analyzing enumeration of colony-forming units (CFUs) following co-culture with MethoCult H4434 Classic (Stem Cell Technologies) and 24-well plates were used to culture cells at a density of 2×10³ for a duration of 14 days. The evaluation of colony-forming unit erythroid (CFU-E), burst-forming unit erythroid (BFU-E), colony-forming unit granulocyte-macrophage (CFU-GM), and colony-forming unit granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM) measurements were conducted using an inverted light microscope (Supplemental Fig. 1). To counteract the effects of TGF-β, supernatants were treated with an anti-TGF-β antibody (clone 1D11, MAB1835, R&D) at a concentration of 1 ug/mL before their combination with CD34 + BM cells and coating for colony assessments as previously describe.

Western blotting

EPCs were collected and disrupted in a lysis buffer (RIPA) supplemented with proteinase inhibitor and PhosSTOP (Fabio science, China). BCA protein assay kit (Fabio science, China) was used to detect protein concentration. The primary antibodies utilized in this investigation were mouse monoclonal antibodies (McAbs) that specifically targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated forms of p38 MAPK, ERK, JNK, c-fos and c-jun as well as the total protein levels of p38, ERK, JNK, c-fos and c-jun (Cell Signaling Technology). The dilutions of these antibodies were prepared following the manufacturer's recommended protocols.

QT-PCR

The extraction of total RNA was performed utilizing the TRIZOL reagent (Takara).The mRNA levels of hematopoietic cytokines and GAPDH were was performed by SYBR green-based RT-qPCR using a RocheLightCycler480 (Roche Molecular Systems, Inc. America). The primers were shown in the Supplemental Table S1.

Enzyme-linked immunosorbent assay (ELISA)

The BM-EPCs with HCMV infection (HCMV Infected group) and without HCMV infection (Normal control group) were cultured in media without serum for an additional 96 hours before collecting the supernatants containing secretome samples (secretome samples). Secretome samples obtained in the absence of cellular presence from BM-EPCs in HCMV Infected group or Normal control group were analyzed for secreted TGF-β1 by ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's recommended protocols.

Samples from human subjects

BM sample was obtained from patients with HCMV viremia (HCMV-emia) accompanied by PGF without antiviral treatment (HCMV(+) + PGF group), patients without HCMV-emia accompanied by PGF without antiviral treatment (HCMV(-) + PGF group), patients without HCMV-emia and good graft function (HCMV(-) + GGF group), patients with HCMV-emia and good graft function (HCMV(+) + GGF group). The patients in above four groups were selected from a shared cohort, ensuring that age, pretransplant conditions, and post-transplant duration were appropriately matched. The four groups did not exhibit any notable distinctions with regards to clinical features, including primary malignancies, CD34 + cell dose transfused for transplantation, and previous episodes of acute graft-versus-host disease (GVHD) (Supplemental Table S2). Healthy individuals were recruited as control subjects (donor group). All subjects were obtained with consent. The present study obtained ethical approval from the Ethics Committee at Nanfang Hospital. The definition of PGF [1–3] is the presence of 2–3 consecutive instances where blood cell counts fall below normal levels (neutrophils ≤ 0.5 × 109/L, platelets ≤ 20 ×109/L, and/or hemoglobin ≤ 70 g/L) for a minimum duration of three days after day + 28 following HSCT or when there is a dependence on transfusions; the presence of hypoplastic-aplastic bone marrow (BM) in cases exhibiting complete donor chimerism; and the absence of concurrent active GVHD or malignancies relapse. HCMV viremia was defined as HCMV-DNA ≥ 500 copies/ml in plasma measured by QT-PCR [21].

Statistical analysis

The data is reported as the average value plus or minus the standard deviation, as specified in the captions of Figs. 1–7. Comparisons among the groups were conducted by employing 1-way ANOVA analysis of variance for statistical analyses. The analyses were conducted utilizing SPSS 25.0 for all statistical computations. The statistical significance was determined at a threshold of P values < .05.

HCMV infects EPCs

The characterization of cultivated BM-EPCs was shown in (Supplemental Fig. 2). To investigate the in vivo infection of BM-EPCs by HCMV in clinical patients, 7-day cultured BM-EPCs from five groups were examined for pp65 expression using IF: donor group, HCMV(-) + GGF group, HCMV(+) + GGF group, HCMV(-) + PGF group, and HCMV(+) + PGF group. The results demonstrated that pp65 expression was exclusively observed in primary BM-EPCs derived from patients in the HCMV(+) + PGF group (Fig. 1A). Conversely, no detectable pp65 expression was found in BM-EPCs obtained from the remaining four groups.

In vitro infection of BM-EPCs with AD169 (HCMV Infected group) resulted in a substantial proportion of cells (> 90%) exhibiting pp65 expression, while non-infected BM-EPCs showed no detectable pp65 expression (Normal control group). (Fig. 1B).

HCMV impairs the function of BM-EPCs

HCMV effects on proliferation, angiogenesis, and apoptosis of BM-EPCs

In BM-EPCs derived from clinical patients and cultured for a duration of 7 days, the quantity of cells exhibiting double-positive staining (Fig. 2A), cellular proliferation(Fig. 2B), capacity for tube formation (Fig. 2C), and capacity for migration (Fig. 2D) were significantly lower in the HCMV(+) + PGF group compared to the other four groups, including donor, HCMV(-) + GGF, HCMV(+) + GGF and HCMV(-) + PGF group. Additionally, the apoptosis level of were significantly lower in the HCMV(+) + PGF group compared to the other four groups (Fig. 2E).

Further evaluation of BM-EPCs function in infection experiments by CMV in vitro showed that BM-EPCs in HCMV Infected group had significantly decreased the proliferation rate (Fig. 2G), tube formation (Fig. 2H) and migration (Fig. 2I) compared with BM-EPCs in Normal control group. Additionally, infected BM-EPCs exhibited an elevated level of apoptosis (Fig. 2J).

HCMV effects on hematopoietic support capacity of BM-EPCs

Hematopoietic support capacity of BM-EPCs was determined by efficiency of CFU plating for CD34 + BM cells following their co-culture with BM-EPCs. In BM-EPCs derived from clinical patients and cultured for a duration of 7 days, the co-cultured CD34 + BM cells in the HCMV(+) + PGF group exhibited significantly lower CFU plating efficiency, as determined by decreased numbers of CFU-E, BFU-E, CFU-GM, and CFU-GEMM, compared to those in the other four groups. (Fig. 2F)

After in vitro CMV infection, the cocultured CD34 + BM cells in the HCMV Infected group exhibited significantly lower CFU plating efficiency compared to those in the Normal control group, as determined by decreased numbers of CFU-E, BFU-E, CFU-GM, and CFU-GEMM. (Fig. 2K).

HCMV suppresses hematopoietic support capacity via secretion of TGF-β1

The expression of hematopoietic-related cytokines in BM-EPCs from clinical patients was assessed using QT-PCR. The results of our study revealed a significant difference solely in the transcription of TGF-β1 among the aforementioned five groups. The transcription level of TGF-β1 in BM-EPCs from the HCMV(+) + PGF group exhibited the highest expression compared to the other four groups (Table 1). However, no significant difference was observed among the donor, HCMV(-) + GGF, HCMV(+) + GGF, and HCMV(-) + PGF groups.

Table 1

**Transcription cytokines of primary bone marrow BM-EPCs in patients.**BM-EPCs, endothelial progenitor cells; CMV, cytomegalovirus; PGF, poor graft function; GGF, good graft function; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-6, interleukin-6; TPO, thrombopoietin; SCF, stem cell factor; SDF-1, stromal cell-derived factor-1; TGF-β1, transforming growth factor beta 1. Superscript: on the same line, the same letters in the top corner indicate no significant difference between groups; different letters in the top corner indicate a significant difference between groups.
Gene	2-△△ct					P
Gene	Donor	CMV(-) + GGF	CMV(+) + GGF	CMV(-) + PGF	CMV(+) + PGF	P
G-CSF	0.98 ± 0.03^a	1.02 ± 0.07^a	1.00 ± 0.03^a	1.04 ± 0.09^a	0.95 ± 0.06^a	0.514
GM-CSF	1.04 ± 0.08^a	1.01 ± 0.04^a	1.01 ± 0.04^a	1.03 ± 0.06^a	0.96 ± 0.01^a	0.428
IL-6	0.99 ± 0.02^a	1.00 ± 0.06^a	1.05 ± 0.04^a	1.07 ± 0.07^a	1.11 ± 0.14^a	0.366
TPO	1.01 ± 0.06^a	1.03 ± 0.09^a	1.01 ± 0.04^a	0.98 ± 0.05^a	1.01 ± 0.01^a	0.931
SCF	1.06 ± 0.12^a	0.95 ± 0.02^a	1.04 ± 0.05^a	1.02 ± 0.13^a	0.97 ± 0.04^a	0.527
SDF-1	1.09 ± 0.12^a	1.02 ± 0.14^a	1.00 ± 0.05^a	1.12 ± 0.20^a	0.95 ± 0.06^a	0.240
TGF-β1	1.16 ± 0.09^a	1.03 ± 0.09^a	0.99 ± 0.01^a	1.12 ± 0.15^a	2.71 ± 0.15^b	< 0.001

In vitro infection experiments, we further identified the HCMV-induced secretion of TGF-β1 by ELISA. The data presented in Fig. 3A demonstrated that secretion of TGF-β1 in BM-EPCs could be induced by HCMV. Supernatants were added to healthy donor derived CD34 + BM cells to quantitate hematopoietic colony formation. The data presented in Fig. 3B demonstrated that under identical culture conditions, the supernatants obtained from the HCMV Infected group exhibited a suppressive effect on colony formation compared to those obtained from the Normal control group. To detect the direct influence of HCMV on CD34 + BM cells that led to impairment of CFU plating efficiency of CD34 + BM cells, CD34 + BM cells were also mixed with serum-free media containing the same copies of HCMV as secretome samples from BM-EPCs in HCMV infected group (HCMV only group). The result showed that no significant reduction CFU plating efficiency of CD34 + BM cells was found in HCMV only group (Fig. 3B). These results indicate that HCMV might suppress hematopoiesis by altering cytokines production of BM-EPCs rather than direct injury of CD34 + BM cells.

To elucidate the potential impact of HCMV-induced TGF-β1 on hematopoietic colony formation inhibition, cell-free supernatants obtained from HCMV Infected group and Normal control group was supplemented with a neutralizing antibody against TGF-β1 at a concentration of 1 µg/ml. The data presented in Fig. 3B demonstrated that after the introduction of a neutralizing antibody against TGF-β1, the levels of total hematopoietic colony formation in the HCMV-Infected group were restored to those observed in the Normal control group.

Phospho-p38 MAPK and phospho-c-fos were elevated in HCMV-infected BM-EPCs

To investigate the impact of MAPK pathways on impaired BM-EPCs in vitro, the levels of p38, ERK and JNK in BM-EPCs were detected by western blot. After infected by HCMV in vitro, BM-EPCs infected by HCMV displayed higher level of phospho-p38 than normal control (Fig. 4A). In contrast, total p38, total ERK, total JNK, and phospho-JNK levels did not exhibit a significant disparity between the BM-EPCs with and without HCMV infection (Fig. 4A).

Western blot technique was utilized to investigate the downstream effects of phospho-p38, specifically AP-1 (including c-fos and c-Jun), which also functions as a regulator for TGF-β1. Significantly increased levels of phospho-c-fos expression were observed in BM-EPCs with HCMV infection (Fig. 4B) than those without HCMV infection. However, there were no notable disparities observed in the levels of intracellular phospho-c-Jun expression between BM-EPCs with and without HCMV infection. (Fig. 4B).

p38 inhibitor and c-fos inhibitor similarly affected the secretion of TGF-β1 of BM-EPCs with HCMV infection in vitro

We investigated the impact of SB203580 inhibition on the functions of infected EPCs in vitro. SB203580 could inhibit the level of phospho-p38 expression (Fig. 4C). In HCMV-infected EPCs, addition of SB203580 could decrease the secretion of TGF-β1 (Fig. 4D) and increase CFU plating efficiency in co-cultured CD34⁺ BM cells (Fig. 4E) determined by increased number of CFU-E, BFU-E, CFU-GM, and CFU-GEMM.

Additionally, the expression of phospho-c-fos was significantly downregulated in HCMV-infected EPCs upon treatment with either SB203580 or the c-fos inhibitor (T-5224). However, addition of T-5224 had no significant effect on expression of phospho-p38 (Fig. 4C). In HCMV-infected EPCs, addition of T-5224 could also decrease the secretion of TGF-β1 (Fig. 4D) and increase CFU plating efficiency in co-cultured CD34⁺ BM cells (Fig. 4E) determined by increased number of CFU-E, BFU-E, CFU-GM, and CFU-GEMM. SB203580 and T-5224 similarly affected the secretion of TGF-β1 and hemopoietic support capacity of BM-EPCs with HCMV infection (Fig. 4D-E).

Our current study demonstrates that HCMV could infect EPCs, resulting in the functional impairments of EPCs. The initiation of p38 MAPK signaling pathway and its subsequent activation of the transcription factor AP−1, induced by HCMV infection, plays a vital role in regulating the secretion of TGF-β1 in BM-EPCs.

Emerging evidence demonstrates that the EPCs abnormality is associated with PGF development post-transplantation [2], but the mechanism of EPCs impairments in PGF patients is not clear. Clinical data indicates that HCMV infection is an important risk factor for PGF [4, 5]. However, no previous study has focued on the impact of CMV on BM-EPCs.

Shi et al. proved the dysfunction of BM-EPCs were found in patients with PGF, which encompased alterations in their proliferative capacity, migratory potential, susceptibility to apoptosis, and ability to promote angiogenesis [2]. Our findings further substantiated the presence of compromised functionality in BM-EPCs following HCMV infection, consistent with the aforementioned dysfunctional BM-EPCs in PGF. Notably, our results showed that the dysfunction observed in BM-EPCs derived from patients with HCMV-emia accompanied by PGF were more pronounced compared to those derived from patients without HCMV-emia accompanied by PGF, indicating the pivotal role of HCMV in the pathogenesis of BM-EPCs injury.

The process of hematopoiesis is precisely regulated through the action of hematopoietic cytokines [20]. Our study revealed that HCMV-infected BM-EPCs exhibited excessive secretion of TGF-β1, a kind of hematopoietic inhibitory factor, which effectively impedes the growth and proliferation of CD34 + hematopoietic progenitor cells (HPCs) [7, 22, 23]. Moreover, we observed that the introduction of a neutralizing antibody against TGF-β1 effectively reversed this suppression of hematopoiesis caused by HCMV infection, demonstrating that the crucial role of TGF-β1 in mediating the suppressive effects on hematopoiesis induced by HCMV.

Shi et al [2] proved that the crucial involvement of p38 MAPK activation in the dysfunction of BM-EPCs observed in patients with PGF, highlighting the pivotal role of MAPKs in the pathogenesis of PGF. AP−1 transcription complex, including fos and jun is one of the most important substrates of MAPK [17]. Previous study demonstrated that AP−1 was highly active following HCMV reactivation and was a determinant of HCMV reactivation [21]. In addition, activation of the AP−1 transcription complex, including fos and jun, contributes to TGF-β1 expression [18, 19, 24]. Therefore, we were specifically interested in the question of whether HCMV caused AP−1-indepent induction of TGF-β1 expression is mediated via the p38 MAPK pathway. Our results showed that the activation of phospho-p38 was not significantly affected by the administration of T−5224 in HCMV-infected BM-EPCs, while addition of SB203580 significantly downregulated the activation of phospho-c-fos, which demonstrated that phospho-c-fos was the downstream target of phospho-p38. Addition of either SB203580 or T−5224 could restores secretion of TGF-β1 and myelopoiesis to levels observed with BM-EPCs in Normal control group. These results demonstrated that HCMV induced increased TGF-β1 secreted by BM-EPCs mainly through MAPK p38-AP−1(c-fos) pathway.

In summary, HCMV could infect BM-EPCs and lead to their dysfunction. Enhanced secretion of TGF-β1 by BM-PECs is induced by HCMV through the up-regulation of p38 MAPK and its downstream transcription factor AP-1, resulting in myelosuppression, which might make a substantial contribution to the pathogenesis of PGF after allo-HSCT, providing innovative therapeutic strategies targeting PGF.

Authors’ degree

Weiran Lv: Doctor of Medicine

Ya Zhou: Master of Medicine

Ke Zhao: Doctor of Medicine

Li Xuan: Doctor of Medicine

Fen Huang: Doctor of Medicine

Zhiping Fan: Doctor of Medicine

Yuan Chang: Master of Medicine

Zhengshan Yi: Doctor of Medicine

Hua Jin: Doctor of Medicine

Yang Liang: Doctor of Medicine

Qifa Liu: Doctor of Medicine

Disclosure

The authors of this manuscript have no conflicts of interest to disclose.

Acknowledgments

This work was supported by Basic and Applied Basic Research Foundation of Guangdong Province, 2022A1515110503.

Ethical approval

The study was approved by the ethics committee of Nanfang Hospital, and written informed consent was obtained from all subjects, in compliance with the Declaration of Helsinki.

Author contributions

Contribution: Q.-F.L., Y.L. and H.J.: designed the study and supervised the manuscript preparation; W.-R.L., Y.Z. and K.Z.: performed the research, analyzed the data, and wrote the manuscript; W.-R.L. and Y.Z.: experimental verification; L.X., F.H. and Z.-P.F.:formal analysis and investigation; Y.C. and Z.-S.Y.: methodology.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Cytomegalovirus Results in Poor Graft Function via Bone Marrow-Derived Endothelial Progenitor Cells

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Abstract

Figures

Introduction

Materials and methods

Viruses

Isolation, cultivation and identification of BM-EPCs

HCMV infects EPCs and HCMV detection

Assessment of cell proliferation, apoptosis and angiogenesis of BM-EPCs

Coculture of BM CD34 + cells with EPCs

Western blotting

QT-PCR

Enzyme-linked immunosorbent assay (ELISA)

Samples from human subjects

Statistical analysis

Results

HCMV infects EPCs

HCMV impairs the function of BM-EPCs

HCMV effects on proliferation, angiogenesis, and apoptosis of BM-EPCs

HCMV effects on hematopoietic support capacity of BM-EPCs

HCMV suppresses hematopoietic support capacity via secretion of TGF-β1

Phospho-p38 MAPK and phospho-c-fos were elevated in HCMV-infected BM-EPCs

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1