Fig 1.
Hematopoietic Stem and Progenitor Cell Populations (HSPC) are vital to vascular endothelial repair and regeneration.
HSPC are CD34+ cells derived from the bone marrow, where they reside in the stromal layer until mobilized in response to chemokines and cytokines released from dysfunctional endothelium. In the peripheral blood, they are capable of differentiating into endothelial progenitor cells (EPC) that will home into cites of vascular dysfunction. The EPC retains the hematopoietic surface marker CD34 in addition to gaining the vascular endothelial cell surface marker CD309 or VEGFR2. These cells will differentiate into mature and circulating endothelial cells capable of incorporating into sites of compromised vasculature, and inducing neovascularization as well as proliferation of existing endothelial cells.
Table 1.
Demographic and hematological characteristics.
Fig 2.
CD34+ cell populations decreased in malaria.
(A) CD34+ hematopoietic stem and progenitor cell populations are significantly decreased in malaria patients. Median fluorescence intensity; 0.5 (IQR 0.3–0.9) in non-malaria, n = 8, vs. 0.2 (IQR 0.1–0.3) in malaria patients, n = 34, p = 0.0006. (B) Circulating EPC numbers (CD45-CD34+VEGFR2+) are decreased in malaria patients by 1.7-fold compared with non-malaria. Median fluorescence intensity; 0.3 (IQR 0.1–0.7) in non-malaria, n = 8 vs malaria 0.1(IQR 0.1–0.2), n = 34, p = 0.02. Data represented as median frequency and Interquartile Range (IQR). Mann-Whitney tests were used to calculate p-value.
Fig 3.
Plasma heme and HO-1 levels increase in malaria patients.
(A) Malaria patients have increased expression of plasma heme (p < 0.0001) and (B) Heme Oxygenase-1 (HO-1) (p < 0.0001) compared to non-malaria subjects. Box plots representing medians with 25th and 75th percentiles, bars for 10th and 90th percentiles, and points for outliers of biomarker concentrations. Means indicated by (+) sign. Statistically significant p-values after Bonferroni adjustment are shown, n = 411. Normal range of 0–60 μM heme and 0–4 ng/mL HO-1 observed in non-malaria subjects.
Fig 4.
Malaria patients have increased expression of TLR4 and plasma CXCL10.
(A) TLR4 Expression is increased in EPC of malaria patients: Median Fluorescence Intensity in non-malaria subjects, 33.7 vs malaria patients, 40.9, p = 0.04. The EPC population was defined as being CD45-CD34+CD309+, (non-malaria n = 8, malaria n = 34). (B) Plasma CXCL10 is significantly increased in malaria patients compared to non-malaria subjects (non-malaria subjects, 178.6 pg/mL (IQR 100.7–333.4), malaria patients, 698.3 pg/mL (IQR 453.8–1143), p < 0.0001). Normal range of 49–811 pg/mL in non-malaria subjects.
Table 2.
Plasma Heme, Heme-Oxyenase-1 and CXCL10 quantification.
Fig 5.
Heme induces apoptosis in HBVEC and CD34+-HSPC in vitro.
Apoptosis was quantified using Guava TUNEL assay and analyzed by fluorescence-activated cell sorting (FACS). Apoptosis was analyzed using analysis of variance followed by Tukey’s multiple comparisons test; In HBVEC p < 0.0001 in heme-treated versus NaOH vehicle, and in CD34+-HSPC p = 0.0004 in heme-treated versus NaOH vehicle. CPT is a potent inducer of apoptosis used as positive control (p < 0.0001 in both cell types).
Fig 6.
Heme mediates TLR expression in vitro.
TLR4 mRNA expression was analyzed using student’s t-test. In HBVEC p = 0.002 in heme-treated versus NaOH vehicle and in CD34+-HSPC p = 0.005 in heme-treated versus NaOH vehicle. Data reported as mean fold-change relative to GAPDH.
Fig 7.
Heme-mediated stimulation of CXCL10 expression is TLR4 dependent.
(A) CXCL10 mRNA expression is TLR4 dependent in HBVEC and CD34+-HSPC in vitro. CXCL10 mRNA expression was analyzed using analysis of variance followed by Tukey’s multiple comparisons test. In HBVEC and CD34+-HSPC, heme-treatment increased CXCL10 expression compared to vehicle (p < 0.0001 and 0.02, respectively). CXCL10 mRNA expression was decreased in the presence of anti-CD14 compared with heme alone (p = 0.0003 and p = 0.11, respectively). Data reported as mean fold-change relative to GAPDH. (B) CXCL10 expression is TLR4 dependent in supernatants of HBVEC and CD34+-HSPC in vitro. CXCL10 protein expression was analyzed using analysis of variance followed by Tukey’s multiple comparisons test. In HBVEC and CD34+-HSPC, heme-treatment increased CXCL10 expression compared to vehicle (p = 0.04 and 0.04, respectively). CXCL10 expression was decreased in the presence of TAK-242 compared with heme alone (p = 0.01, and 0.03, respectively). The 2(-Delta Delta C(T)) method was used to analysis relative gene expression data normalized to housekeeping gene, GAPDH, which was unaffected by experimental. Results are expressed as fold change relative to housekeeping gene in treatment versus vehicle-treated cultures.