Chemokine Ligand 5 (CCL5) Derived from Endothelial Colony-Forming Cells (ECFCs) Mediates Recruitment of Smooth Muscle Progenitor Cells (SPCs) toward Critical Vascular Locations in Moyamoya Disease

Abstract

The etiology and pathogenesis of moyamoya disease (MMD) are still obscure. Previous studies indicated that angiogenic chemokines may play an important role in the pathogenesis of the disease. Recently, it was discovered that peripheral blood-derived endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SPCs) have defective functions in MMD patients. Therefore, the interaction of ECFCs and SPCs, the precursors of two crucial cellular components of vascular walls, with some paracrine molecules is an intriguing subject. In this study, co-culture of ECFCs and SPCs from MMD patients and healthy normal subjects revealed that MMD ECFCs, not SPCs, are responsible for the defective functions of both ECFCs and SPCs. Enhanced migration of SPCs toward MMD ECFCs supported the role for some chemokines secreted by MMD ECFCs. Expression arrays of MMD and normal ECFCs suggested that several candidate cytokines differentially produced by MMD ECFCs. We selected chemokine (C-X-C motif) ligand 6 (CXCR6), interleukin-8 (IL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 for study, based on the relatively higher expression of these ligands in MMD ECFCs and their cognate receptors in MMD SPCs. Migration assays showed that only CCL5 significantly augmented the migration activities of SPCs toward ECFCs. Treatment with siRNA for the CCL5 receptor (CCR5) abrogated the effect, confirming that CCL5 is responsible for the interaction of MMD ECFCs and SPCs. These data indicate that ECFCs, not SPCs, are the major players in MMD pathogenesis and that the chemokine CCL5 mediates the interactions. It can be hypothesized that in MMD patients, defective ECFCs direct aberrant SPC recruitment to critical vascular locations through the action of CCL5.

Fig 1. In vitro tube formation assays (original magnification ×100).

(A and B) MMD ECFCs (green colored) make fewer tubes per unit area than normal ECFCs (n = 6, 11.9 ± 4.0% of control; p<0.001). MMD SPCs (red colored) also make less number of tubes (n = 6, 51.8 ± 21.5% of control; p<0.001). (C) The tube walls made by MMD ECFCs are thinner (n = 5, 14.4% of control; p< 0.001), but the tube walls composed of MMD SPCs are thicker than those of controls (n = 5, 763.4% of control; p<0.001) (*p<0.05, **p<0.01, ***p<0.001).

Fig 2. Co-culture experiments of ECFCs (green colored) and SPCs (red colored) (original magnification ×100).

(A) In co-culture system (n = 7 for each group), both ECFCs and SPCs contribute to the tube formation. (B and C) MMD ECFCs when co-cultured with either normal SPCs or MMD SPCs make significantly less number of tubes than normal ECFCs (p = 0.168 for [normal ECFCs + normal SPCs] vs. [normal ECFCs + MMD SPCs]; p<0.001 for [normal ECFC+ normal SPC] vs. [MMD ECFC+ normal SPC]; p<0.001 for [normal ECFC+ normal SPC] vs. [MMD ECFC+ MMD SPC]).

Fig 3. Trans-well migration assays (original magnification ×200).

Migration of SPCs is enhanced when MMD ECFCs are placed in the bottom well (n = 4 for each group). The cell intensity value for combination of normal SPCs and MMD ECFCs is 166 ± 58.9% of control (combination of normal SPCs and normal ECFCs) (p = 0.029). The cell intensity for combination of MMD SPCs and MMD ECFCs are 539 ± 161.8% of control (p<0.001).

Fig 4. Differentially expressed chemokines in MMD ECFCs.

(A) Expression of various chemokines are compared between normal ECFCs (n = 4) and MMD ECFCs (n = 7). (B) KEGG pathway enrichment analysis; upregulated genes are involved in chemokine signaling pathway. (C) Four most-up-regulated chemokines in MMD ECFCs are selected for further analysis. (D) Confirmation of mRNA expression of select gene in ECFCs by RTq-PCR (n = 4 for each group). Significantly higher levels of CLCL6, IL8, and CCL5 mRNA are expressed in MMD-ECFCs (CXCL6, p = 0.038; IL8, p = 0.021; CCL2 p = 0.239; CCL5, p = 0.008).

Fig 5. Augmentation of SPCs migration by rhCCL5 (original magnification ×200).

(A and B) Addition of rhCCL5, not of CXCL6 or IL8 enhances the migration of MMDSPCs across the trans-well membranes without ECFCs in the bottom wells (n = 3 for each group). A dose-dependent increase of migration is observed (p = 0.027). (C) There is no difference in cell viability with the addition of rhCXCL6, rhIL8 or rhCCL5 (n = 5 for each group).

Fig 6. Blocking CCL5-CCR5 interaction with siRNA (original magnification ×200).

(A) In trans-well migration assays (n = 3 for each group), treatment of CCR5 siRNAs effectively abrogates the enhanced migration of MMD SPCs by the presence of MMD ECFCs or addition of rhCCL5. (B) Quantification of migrated cells.