Atorvastatin enhances bone marrow endothelial cell function in corticosteroid-resistant immune thrombocytopenia patients

Immune thrombocytopenia (ITP), generally characterized by increased peripheral platelet destruction and reduced platelet production, is a common hematological disease.^1-6  A panel of the International Working Group (IWG) prefers “immune” to emphasize the immune-mediated mechanism of ITP.⁷  Moreover, corticosteroids represent the standard first-line therapy, achieving responses in ∼50% to 85% of ITP patients.^8-14  However, for the remaining corticosteroid-resistant ITP patients, who exhibit either no response to corticosteroid or are corticosteroid-dependent,^7,8,15  the pathogenesis remains poorly understood and the clinical management is challenging.

Thrombopoiesis, which occurs within a specialized bone marrow (BM) microenvironment, is a complex biological process that is initiated with the commitment of hematopoietic stem cells (HSCs) to megakaryocytic progenitors and eventually results in the maturation of megakaryocytes to produce functional platelets.^16,17  Abnormalities during any stage of megakaryocytopoiesis might influence platelet production. Endosteal cells, endothelial progenitor cells (EPCs), perivascular cells, and various mature immune cells have been revealed to be key elements of the BM microenvironment.^18-23  Emerging evidence from murine studies suggested that BM EPCs might play a crucial role in regulating thrombopoiesis.^24-26  We previously reported that the impaired BM EPCs contribute to the occurrence of prolonged isolated thrombocytopenia (PT), which is a serious complication defined as the engraftment of all peripheral blood cell lines other than platelets (with a count <20 × 10⁹/L) or dependence on platelet transfusions after allogeneic HSC transplantation (allo-HSCT).²⁷  Similar to PT, ITP is characterized by low platelet counts and a maturation disorder of megakaryocytes. However, the functional role of BM EPCs in ITP patients is largely unknown. We recently reported that the number of BM EPCs was normal, but an abnormal BM immune microenvironment was observed in corticosteroid-sensitive ITP patients, indicating that dysregulated T-cell responses in the BM microenvironment might play an important role in the pathogenesis of corticosteroid-sensitive ITP.²⁸  Nevertheless, the number and functional role of BM EPCs in corticosteroid-resistant ITP patients have never been reported. Moreover, approaches to improve the dysfunction of BM EPCs in corticosteroid-resistant ITP patients remain unidentified.

Atorvastatin, a synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is frequently used in patients with dyslipidemia and associated vascular abnormalities.^29,30  Moreover, atorvastatin has been reported to improve the mobilization and function of circulating EPCs in heart disease, diabetes, and other conditions^31-33  by downregulating p38 MAPK^34,35  or upregulating Akt signaling.^36,37  Our recent study suggested that atorvastatin represents a promising therapeutic approach for repairing the impaired BM EPCs in patients with poor graft function (PGF) post allo-HSCT.³⁸  Nevertheless, no previous studies have focused on the roles of atorvastatin in BM EPCs in corticosteroid-resistant ITP patients.

Therefore, the current study was performed to evaluate whether the quantity and function of BM EPCs in corticosteroid-resistant ITP patients differed from those in corticosteroid-sensitive ITP patients. Moreover, we investigated the effects of atorvastatin on the number and function of the cultivated BM EPCs derived from corticosteroid-resistant ITP patients and their underlying molecular mechanisms. Subsequently, a pilot study was performed to evaluate the efficacy and safety of atorvastatin in corticosteroid-resistant ITP patients. Our aim was to provide new insights into the pathogenesis underlying corticosteroid-resistant ITP.

In the prospective case-control study, corticosteroid-resistant ITP patients (N = 55) and their matched corticosteroid-sensitive ITP patients (N = 55) were identified from the same cohort of primary ITP patients from 1 September 2016 to 31 August 2017, and from 1 October 2017 to 23 November 2017, at Peking University Institute of Hematology (supplemental Table 1, available on the Blood Web site). The 2 groups of ITP patients were matched for age, sex, and the time at which the BM microenvironment was evaluated after corticosteroid therapy (±3 days; “risk-set sampling”).³⁹ 

BM samples from transplant donors (N = 30) were considered as healthy controls. The healthy cohort comprised 11 men and 19 women, aged 18 to 55 years (median, 42 years). The study was approved by the ethics committee of Peking University People’s Hospital, and written informed consent was obtained from all subjects, in compliance with the Declaration of Helsinki.

From 1 March 2017 to 31 August 2017, after written consent in compliance with the Declaration of Helsinki, 13 of the 18 patients with corticosteroid-resistant ITP,⁸  who were willing to accept oral treatment with atorvastatin (20 mg per day) and N-acetyl-l-cysteine (NAC; a reactive oxygen species [ROS] scavenger; 400 mg 3 times a day), were enrolled in the pilot study. As shown in supplemental Table 2, patients who were receiving other maintenance regimens, including corticosteroids and danazol, were also eligible if the treatment dose had been stable in the past month and the dose was expected to remain unchanged or taper off at least the first 4 weeks of the study until the initial response was evaluated.

The primary end points of the pilot study were complete response (CR), response, and overall response (OR). Secondary end points were time to response and adverse events (safety). The treatment responses were evaluated weekly after the interventions. The definitions of the treatment responses were as follows: (1) CR was defined as a platelet count of at least 100 × 10⁹/L; (2) response was defined as a platelet count between 30 × 10⁹/L and 100 × 10⁹/L and at least doubling of the baseline count; (3) OR included CR and response; and (4) no response (NR) was defined as a platelet count lower than 30 × 10⁹/L or less than doubling of the baseline count.⁷ 

According to the IWG guidelines,^7,8  primary ITP was defined as an autoimmune disorder characterized by isolated thrombocytopenia (peripheral blood platelet count <100 × 10⁹/L) in the absence of other causes or disorders that might be associated with thrombocytopenia (eg, BM failure or myelodysplastic states, hepatitis C, or Helicobacter pylori infection). Corticosteroid-resistant ITP was defined as no response to at least 1 full-dose corticosteroid, defined as prednisone 1 mg/kg daily for at least 4 weeks then tapered off,^7,8,15  to maintain a platelet count at or above 30 × 10⁹/L and/or to avoid bleeding, or depending on the corticosteroid.^7,8,15  Corticosteroid-sensitive ITP was defined as maintaining a platelet count at or above 30 × 10⁹/L and/or to avoid bleeding after the initial corticosteroid treatment.^7,8 

The quantity and function of BM EPCs from the corticosteroid-resistant ITP patients and their matched corticosteroid-sensitive ITP patients were evaluated after 4 weeks of corticosteroid treatment. The numbers of patients performed for the independent experiments were labeled in each figure. In the pilot study, BM EPCs were evaluated pre- and posttreatment only in the patients who were willing to provide BM samples after written consent.

Isolation, cultivation, and characterization of BM EPCs were performed as previously reported.³⁸  In brief, BM mononuclear cells (BMMNCs) were cultivated with EGM-2-MV-SingleQuots (Lonza, Walkersville, MD) and 10% fetal bovine serum (Gibco, Rockville, MD) at 37°C in a humidified incubator with 5% CO₂ for 7 days before being used in experiments in 24-well culture plates (Corning Incorporated, Corning, NY) that were precoated within fibronectin (Sigma, St. Louis, MO).

Precultivated and 7-day cultivated BM EPCs were identified by staining with mouse anti-human CD34, vascular endothelial growth factor receptor 2 (VEGFR2; CD309), and CD133 monoclonal antibodies (BD Biosciences, San Jose, CA). The samples were then evaluated using LSRFortessa (Becton Dickinson). Aliquots of isotype-identical antibodies served as negative controls.

The cultivated adherent cells were incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil)–acetylated low-density lipoprotein (DiI–Ac-LDL; Life Technologies, Gaithersburg, MD) and fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (FITC–UEA-I; Sigma).³⁸  To evaluate the numbers of double-positive–stained EPCs per well, 3 random power fields were counted using a fluorescence microscope (Olympus, Tokyo, Japan).

As previously reported,^38,40  the cell counting, cell proliferation assay, migration assay, tube-formation assay, and apoptosis analysis were analyzed in BM EPCs after treatment with: 50 nM, 500 nM, 5000 nM atorvastatin (Sigma); 0.1 mM, 1 mM, 10 mM NAC (Sigma); atorvastatin (500 nM) combined with NAC (1 mM); 10 μM p38 inhibitor (SB203580; Sigma); and 400 nM p38 inhibitor (BIRB796; Selleck Chem, Houston, TX) for 24 hours.

The cells stained with the aforementioned EPC markers were incubated with 10 μM 2′,7′-dichlorofluorescence diacetate (Byotimes, Beijing, China) at 37°C for 20 minutes. The mean fluorescence intensity of intracellular ROS was measured on a LSRFortessa (Becton Dickinson).³⁸ 

As previously reported,³⁸  cells were incubated with EPC antibodies at 4°C for 20 minutes and then fixed, permeabilized, and incubated with the following antibodies: phospho-p38 (p-p38), phospho–extracellular signal-regulated kinase (p-ERK), phospho-JNK (p-JNK) (Cell Signaling Technology, Danvers, MA), and phospho-Akt (p-Akt; Becton Dickinson). The intracellular protein levels were evaluated by LSRFortessa software (Becton Dickinson) and expressed as the mean fluorescence intensity (means ± standard error of the mean [SEM]).

Cultivated EPCs were washed and incubated in radioimmunoprecipitation assay buffer containing proteinase inhibitor and PhosSTOP (Roche, Indianapolis, IN) as described previously.³⁸  The protein levels were determined using a BCA kit (Thermo Scientific, Rockford, IL). The proteins (30-50 µg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (Millipore, Bedford, MA) membranes. The membranes were then blocked with 5% bovine serum albumin and incubated overnight with antibodies against p-p38 MAPK, total p38, p-ERK, total ERK, p-JNK, total JNK, p-Akt, total Akt, and actin (Cell Signaling Technology) at dilutions specified by the manufacturer’s instructions. Anti-rabbit or anti-mouse secondary antibodies and an ECL chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL) were used according to the manufacturer’s instructions to scan and semiquantitatively analyze the proteins.

BM trephine biopsies (BMBs) were obtained from the posterior superior iliac spine, and fixed with 4% paraformaldehyde and processed for frozen sections as previously described.⁴¹  After equilibrium at room temperature (RT) for 30 minutes, nonspecific antibody binding was blocked by incubating the slides with 20% goat serum at RT for 1 hour. Mouse anti-human CD34 (Becton Dickinson) and rabbit anti-human CD133 (Abcam, Cambridge, MA) were incubated at 4°C overnight. Goat anti-rabbit 555 and donkey anti-mouse 488 (Invitrogen, Eugene, OR) were added at RT for 1 hour. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the nucleus, and the prepared slides were analyzed using Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany).

Colony-forming units (CFUs) were assayed using MethoCult H4434 Classic (Stem Cell Technologies, Vancouver, BC, Canada) as described previously.^38,40  After 7 days of cultivation, suspended CD34⁺ cells were harvested, and 5 × 10⁴ cells were plated and cultured for 14 days. 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 scored on an inverted light microscope. Cultures were assayed in duplicate, and the results are expressed as the means ± SEM.

BM EPCs from patients with corticosteroid-resistant ITP, or corticosteroid-sensitive ITP, were cultivated for 7 days. Then, CD34⁺ cells (1 × 10⁵ cells per well) were isolated from BMMNCs of healthy donors with a CD34 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and placed in a direct contact culture with 1 mL of StemSpan SFEM (Stem Cell Technologies) containing 100 ng/mL stem cell factor, 100 ng/mL thrombopoietin, and 10 ng/mL interleukin-3 for another 7 days for megakaryocyte differentiation.

Colony-forming unit megakaryocytes (CFU-MKs) were counted with a commercially available kit (MegaCult-C; Stem Cell Technologies) according to the manufacturer’s instructions. Cocultured CD34⁺ cells were plated in chamber slides (5 × 10⁴ cells per well) and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 10 to 12 days. After dehydration, fixation, and immunocytochemical staining with mouse anti–human glycoprotein IIb/IIIa antibody and biotin-conjugated goat anti–mouse immunoglobulin G, megakaryocyte colonies were defined as groups of 3 or more glycoprotein IIb/IIIa⁺ cells.

Quantification of the megakaryocytes, megakaryocyte apoptosis, and platelets produced in the cultures were performed as previously described.^42,43 

Statistical analyses were performed using 1-way analysis of variance for comparisons among the groups. Subject variables were compared using the χ² test for categorical variables and the Mann-Whitney U test for continuous variables. The Wilcoxon test for paired data were used to identify the drug effects. Analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA), and P values <.05 were considered significant.

As shown in supplemental Table 1, age, sex, white blood cell count, and hemoglobin showed no significant differences between the 2 groups of ITP patients when the BM microenvironment was evaluated. However, the platelet count in corticosteroid-resistant ITP was significantly lower than those in corticosteroid-sensitive ITP. More corticosteroid-resistant ITP patients received danazol and cyclosporine A as prior and concomitant therapies compared with corticosteroid-sensitive ITP patients when the BM microenvironment was evaluated.

Baseline percentages of BM EPCs (Figure 1A; 0.02% ± 0.003% vs 0.12% ± 0.04%; P = .0001) from corticosteroid-resistant ITP were significantly lower than those in corticosteroid-sensitive ITP. The levels of ROS (Figure 1B; 1214 ± 122 vs 591.2 ± 45.4; P < .0001) and apoptosis (Figure 1C; 33.1% ± 3.6% vs 22.4% ± 4.3%; P = .02) of precultured BM EPCs from corticosteroid-resistant ITP were significantly higher than those from corticosteroid-sensitive ITP.

Consistent with our flow cytometry data, in situ immunofluorescent staining with CD34 and CD133 revealed that the quantification of BM CD34⁺CD133⁺ EPCs was significantly reduced in corticosteroid-resistant ITP compared with those in the corticosteroid-sensitive ITP and the healthy controls (Figure 1D).

At day 7 of cultivation, the typical EPC phenotype³⁸  was confirmed by demonstrating the positive expression of CD34, CD309, and CD133 by flow cytometry (Figure 2A). Moreover, the spindle-shaped and elongated BM adherent cells were further functionally characterized as EPCs by their capacities of DiI–Ac-LDL uptake and FITC–UEA-I binding (Figure 2B).

To evaluate the quantity and function of primary BM EPCs, cell count, double-positive staining of DiI–Ac-LDL and FITC–UEA-I, and angiogenic potential (including migration and tube formation) were analyzed.

At day 7 of cultivation, double-positive–stained BM EPCs from corticosteroid-resistant ITP patients were significantly lower than those in corticosteroid-sensitive ITP (Figure 2B; 5.5 ± 0.9 vs 17 ± 2.6; P = .0001).

Moreover, 7-day cultivated BM EPCs from corticosteroid-resistant ITP showed significantly reduced tube formation (Figure 2C; 3514 ± 437 vs 6268 ± 800.7; P = .02) and migration (Figure 2D; 86.2 ± 5.6 vs 148.8 ± 14.8; P < .0001).

Notably higher levels of p-p38 (Figure 3A; 10 182 ± 1022 vs 4917 ± 504.5; P = .0001) and lower levels of p-Akt (Figure 3B; 581.4 ± 60.7 vs 1188 ± 247.8; P = .006) were detected in BM EPCs from corticosteroid-resistant ITP patients than in those from corticosteroid-sensitive ITP patients. In contrast, the levels of intracellular p-ERK (Figure 3C; 6141 ± 416.8 vs 6805 ± 943.9; P = .57) and p-JNK (Figure 3D; 1456 ± 162.3 vs 1703 ± 192.5; P = .57) were not significantly different between corticosteroid-resistant ITP and corticosteroid-sensitive ITP. Similarly, the basal expression patterns of these intracellular proteins detected by flow cytometry were further validated in cultivated BM EPCs by western blot (Figure 3E).

We first investigated the dose-response effect and found that atorvastatin (500 nM) and NAC (1 mM) demonstrated superior effects on the cultivated BM EPCs from corticosteroid-resistant ITP patients (supplemental Figure 1).

Atorvastatin (500 nM) or NAC (1 mM) significantly increased the quantity of BM EPCs, including the double-positive–stained cells (Figure 4A; 2.5-fold ± 0.3-fold, P = .004; 2.2-fold ± 0.4-fold, P = .02) and total number (Figure 4D; 2.3-fold ± 0.4-fold, P = .003; 2.3-fold ± 0.5-fold, P = .04) of BM EPCs, in corticosteroid-resistant ITP compared with untreated cells. Moreover, atorvastatin or NAC ameliorated the function of BM EPCs in corticosteroid-resistant ITP patients, including tube-formation capability (Figure 4B; 1.8-fold ± 0.3-fold, P = .047; 2.2-fold ± 0.4-fold, P = .02), migration capacity (Figure 4C; 1.4-fold ± 0.2-fold, P = .04; 1.3-fold ± 0.2-fold, P = .04), and proliferation rate (Figure 4F; 1.3-fold ± 0.1-fold, P = .02; 1.3-fold ± 0.1-fold, P = .004). Reduced levels of intracellular ROS (Figure 4E; 0.8-fold ± 0.1-fold, P = .004; 0.8-fold ± 0.1-fold, P = .002) and apoptosis (Figure 4G; 0.8-fold ± 0.1-fold, P = .004; 0.8-fold ± 0.1-fold, P = .03) were observed in atorvastatin or NAC treatment groups.

Furthermore, in vitro treatment with atorvastatin or NAC slightly influenced the number and function of BM EPCs from corticosteroid-sensitive ITP patients (Figure 4). These data suggest that BM EPCs from corticosteroid-resistant ITP patients are more sensitive to atorvastatin or NAC than those from corticosteroid-sensitive ITP patients.

Treatment with NAC and p38 inhibitors (SB203580 and BIRB796) increased the percentages (Figure 5A; 1.6-fold ± 0.2-fold, P = .006; 1.1-fold ± 0.1-fold, P = .5; 1.3-fold ± 0.2-fold, P = .3), double-positive–stained cells (Figure 5C; 3.6-fold ± 0.8-fold, P = .002; 3.9-fold ± 1.0-fold, P = .02; 3.1-fold ± 0.6-fold, P = .01), proliferation (Figure 5D; 1.5-fold ± 0.2-fold, P = .02; 1.4-fold ± 0.2-fold, P = .08; 1.6-fold ± 0.3-fold, P = .06), and migration (Figure 5F; 1.4-fold ± 0.1-fold, P = .0001; 1.1-fold ± 0.1-fold, P = .01; 1.1-fold ± 0.1-fold, P = .09) of BM EPCs in corticosteroid-resistant ITP patients. NAC and p38 inhibitors reduced ROS (Figure 5B; 0.5-fold ± 0.1-fold; P = .03; 0.5-fold ± 0.1-fold; P = .04; 0.5-fold ± 0.1-fold, P = .0498) and apoptosis levels (Figure 5E; 0.6-fold ± 0.1-fold, P = .002; 0.5-fold ± 0.1-fold, P = .002; 0.5-fold ± 0.1-fold, P = .004) of BM EPCs from corticosteroid-resistant ITP. These findings indicate that inhibiting ROS or p38 activation enhances the number and function of BM EPCs in corticosteroid-resistant ITP patients, similar to atorvastatin treatment.

The higher p-p38 expression in BM EPCs from corticosteroid-resistant ITP patients decreased significantly following treatment with atorvastatin, NAC, p38 inhibitors, or atorvastatin/NAC cotreatment (Figure 5G; 0.8-fold ± 0.1-fold, P = .003; 0.8-fold ± 0.1-fold, P = .01; 0.8-fold ± 0.1-fold, P = .006; 0.7-fold ± 0.1-fold, P = .001; 0.5-fold ± 0.1-fold, P < .0001), whereas p-Akt was increased significantly in the different treatment groups (Figure 5H; 2.5-fold ± 0.3-fold, P = .0002; 2.7-fold ± 0.3-fold, P < .0001; 2.0-fold ± 0.3-fold, P = .04; 2.4-fold ± 0.4-fold, P = .004; 2.9-fold ± 0.5-fold, P = .0005), which is consistent with the western blot analysis results (Figure 5I).

The levels of intracellular ROS in primary BM CD34⁺ cells derived from corticosteroid-resistant ITP patients were significantly higher than those in corticosteroid-sensitive ITP patients (Figure 6B; 4350 ± 415.3 vs 2590 ± 238; P = .0009), whereas no significant differences in the cell counts (Figure 6A) or apoptosis rates (Figure 6C) of BM CD34⁺ cells were found between the 2 groups.

As shown in Figure 6D, the BM CD34⁺ cells from corticosteroid-resistant ITP patients had a deficit in CFU-GEMM (0.6 ± 0.3 vs 2.7 ± 0.3; P = .008) outgrowth at a baseline level compared with corticosteroid-sensitive ITP patients, whereas the CFU-E, BFU-E, and CFU-GM outgrowth showed no significant differences.

Treatment with atorvastatin, NAC, p38 inhibitors, or atorvastatin/NAC cotreatment improved the percentages of megakaryocytes (Figure 6E; 1.6-fold ± 0.2-fold, P = .02; 1.8-fold ± 0.3-fold, P = .04; 1.6-fold ± 0.3-fold, P = .27; 1.5-fold ± 0.3-fold, P = 0.23; 1.8-fold ± 0.2-fold, P = .002), megakaryocyte apoptosis (Figure 6F; 1.2-fold ± 0.1-fold, P = .02; 1.1-fold ± 0.1-fold, P = .02; 1.1-fold ± 0.1-fold, P = .1; 1.1-fold ± 0.1-fold, P = 0.16; 1.4-fold ± 0.1-fold, P < .0001), megakaryocyte ploidy distribution (Figure 6G; 2.1-fold ± 0.3-fold, P = .02; 2.2-fold ± 0.4-fold, P = .02; 1.3-fold ± 0.3-fold, P = 0.87; 1.5-fold ± 0.3-fold, P = .61; 2.6-fold ± 0.4-fold, P = .004). and platelet release (Figure 6H; 2.6-fold ± 0.6-fold, P = .02; 2.7-fold ± 0.7-fold, P = .04; 2.1-fold ± 0.5-fold, P = .35; 2.9-fold ± 0.7-fold, P = .01; 3.4-fold ± 0.6-fold, P = .0002) of BM EPCs from corticosteroid-resistant ITP patients to support CD34⁺ hematopoietic progenitors from healthy donors. Moreover, the CFU-MK–plating efficiency (Figure 6I; 2.6-fold ± 0.6-fold, P = .01; 2.7-fold ± 0.7-fold, P = .02; 2.1-fold ± 0.5-fold, P = 0.23; 3.0-fold ± 0.6-fold, P = .004; 3.6-fold ± 0.7-fold, P < .0001) from the intervention groups were significantly higher than those in the vehicle-treated control group. These data suggest that atorvastatin, NAC, or p38 inhibitors could improve the ability of the impaired BM EPCs from corticosteroid-resistant ITP patients to support HSC differentiation into megakaryocytes and ultimately to platelet production, as determined by the CFU-MK–plating efficiency in vitro. Notably, atorvastatin and NAC provided markedly better repair capacity to BM EPCs from corticosteroid-resistant ITP patients than did the p38 inhibitors.

The CR, response, and OR rates of atorvastatin and NAC treatment were 23.1% (3 of 13), 46.2% (6 of 13), and 69.2% (9 of 13) in corticosteroid-resistant ITP patients. In patients who achieved CR/R, the median time to response was 25 days (7-51 days), and no apparent adverse events were observed. The platelet counts in corticosteroid-resistant ITP patients who achieved CR/response (N = 9) were significantly increased posttreatment compared with pretreatment (Figure 7A; 61 × 10⁹/L vs 21 × 10⁹/L; P = .004).

Compared with the baseline levels before atorvastatin and NAC treatment, the quantity and function of BM EPCs and CD34⁺ cells were improved to some degree in the patients who showed CR/response after treatment when evaluated by flow cytometry and immunofluorescence (Figure 7B-I). By contrast, no significant improvement was found in the NR patients posttreatment when compared with their baseline levels pretreatment (Figure 7B-H).

The current study provided the first demonstration that reduced and dysfunctional BM EPCs, characterized by decreased migration and angiogenesis, as well as higher levels of ROS and apoptosis, were involved in the pathogenesis of corticosteroid-resistant ITP. Moreover, atorvastatin quantitatively and functionally improved BM EPCs derived from corticosteroid-resistant ITP patients by downregulating p38 MAPK and upregulating Akt pathway; atorvastatin also partially rescued the impaired BM EPCs to support megakaryocytopoiesis. Although requiring further validation, our preliminary data suggest that atorvastatin is a promising therapeutic strategy in corticosteroid-resistant ITP patients.

Abnormal megakaryocyte maturation and apoptosis in BM and increased peripheral platelet destruction have been reported to be involved in the occurrence of primary ITP.^1,28,44-49  BM EPCs, which have been identified as a critical element of the BM microenvironment, possess the capacity to support hematopoiesis and megakaryocytopoiesis.^25,50  In this regard, murine studies suggested that the effective cross-talk between EPCs and megakaryocytes regulates megakaryocyte maturation and thrombopoiesis in BM microenvironment.^25,50  Moreover, disruption of the BM EPCs or interference with megakaryocyte motility was found to inhibit thrombopoiesis, both under physiological conditions and after myelosuppression.^24,26  However, no previous study has focused on the number and functional role of BM EPCs in corticosteroid-resistant ITP patients.

BM EPCs were found quantitatively and functionally impaired in corticosteroid-resistant ITP patients, which is consistent with the defective BM EPCs in PT patients post allo-HSCT.²⁷  In contrast, Song et al reported that the frequency of BM EPCs was normal in corticosteroid-sensitive ITP patients, whereas dysregulated T-cell responses in the BM microenvironment were involved in corticosteroid-sensitive ITP.²⁸  These findings might explain why corticosteroid-resistant ITP patients exhibit either no response to corticosteroids or are corticosteroid-dependent, indicating that immune-mediated platelet destruction is not the sole pathogenic mechanism. Considering the predominant role of EPCs in supporting megakaryocytopoiesis in the BM microenvironment,^24,25  as well as the current study, it is conceivable that impaired BM EPCs might hamper the differentiation progress from HSCs to megakaryocytes, resulting in the occurrence of corticosteroid-resistant ITP, which is different from the immune-mediated mechanism in corticosteroid-sensitive ITP.^28,38  Although more corticosteroid-resistant ITP patients received danazol or cyclosporine A as prior and concomitant therapies, no harmful effects were reported about danazol and cyclosporine A on EPC biology.^51,52 Therefore, we speculated that the BM EPCs differences between the 2 groups of ITP patients might be due to their disease biology rather than, for example, treatment effects. Consequently, therapeutic strategies to enhance the prognosis of corticosteroid-resistant ITP patients through improving BM EPCs are urgently needed.

Direct infusion of circulating EPCs can improve endothelial repair, significantly increase BM HSCs, and ultimately accelerate hematopoietic and immune reconstitution after allo-HSCT in mice.⁵³  However, the critical limitation for the therapeutic application of EPC infusion in humans is their low number in circulation. In contrast, atorvastatin showed minimal toxic effects, can be easily administered orally, and is also affordable. We recently reported that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in PGF patients through downregulating the p38 MAPK pathway. Different from our previous study in PGF,³⁸  atorvastatin not only quantitatively and functionally improved BM EPCs from corticosteroid-resistant ITP patients by downregulating p38 MAPK and upregulating Akt pathway, but also partially rescued the impaired ability of BM EPCs to support megakaryocytopoiesis. Although the underlying molecular mechanisms are needed to further explore, the repair of endogenous BM EPCs via exogenous agents represents a potential therapeutic approach in corticosteroid-resistant ITP patients through regulating megakaryocytopoiesis.

We are aware, however, that the pathogenesis of corticosteroid-resistant ITP is heterogeneous. It would be interesting and informative to further investigate whether the impaired BM EPCs directly affect megakaryocytopoiesis or act through their immunoregulatory effects in the occurrence of corticosteroid-resistant ITP. Moreover, atorvastatin might have effects on many other cells in corticosteroid-resistant ITP patients, which needs to be further clarified.

In summary, these results indicate that reduced and dysfunctional BM EPCs play a role in the pathogenesis of corticosteroid-resistant ITP, and the impaired BM EPCs could be improved by atorvastatin. Our preliminary data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in corticosteroid-resistant ITP patients. We are aware, however, that further prospective large-scale randomized clinical trials, particularly in other ethnicities, are needed to validate our findings in the future.

The authors thank all of the core facilities at Peking University Institute of Hematology for sample collection. The authors thank Cai-Wen Duan from Shanghai Jiao Tong University School of Medicine for excellent assistance with the immunofluorescent staining. American Journal Experts (www.journalexperts.com) provided editorial assistance to the authors during the preparation of the manuscript.

This work was supported by the National Natural Science Foundation of China (grant nos. 81570127, 81370638, and 81530046), the Science and Technology Project of the Guangdong Province of China (2016B030230003), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81621001), and the National Key Research and Development Program (2017YFA0104500).

Contribution: X.-J.H. and Y.K. designed the study and supervised the manuscript preparation; Y.K., X.-N.C., and X.-H.Z. performed the research; Y.K. and X.-N.C. analyzed the data and wrote the manuscript; M.-M.S., Y.-Y.L., Y.W., L.-P.X., and Y.-J.C. participated in the collection of patients’ data; and all authors agreed to submit the final manuscript.

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

Correspondence: Xiao-Jun Huang, Peking University People's Hospital, Peking University Institute of Hematology, No. 11 Xizhimen South St, Beijing, 100044, China; e-mail: xjhrm@medmail.com.cn.