Impairment of two types of circulating endothelial progenitor cells in patients with glucocorticoid-induced avascular osteonecrosis of the femoral head

Abstract

Objectives

This study examined whether abnormalities of early EPCs and endothelial colony forming cells (ECFCs) are present and compared their functions in glucocorticoid (GC)-induced avascular osteonecrosis of the femoral head (ANFH).

Methods

Early EPCs and endothelial colony forming cells (ECFCs) were obtained from 33 patients with glucocorticoid-induced ANFH and 33 age- and sex-matched control subjects. Cells were isolated, in vitro cultured and studied by Flow Cytometry and Immunofluorescence. Colony-forming unit counts were observed from 33 patients and 33 healthy controls. Growth kinetics, migratory capacity to multiple chemo-attractants, in vitro tube formation capacity and cytokine (vascular endothelial growth factor and stromal cell-derived factor-1) levels in supernatants of two types of EPCs were assayed in ANFH patients and matched controls (n = 4).

Results

Mean numbers of colonies formed by both types of EPCs were decreased in ANFH patients (Early EPCs: 2.42 ± 1.46 versus 4.52 ± 2.00, p < 0.05; ECFCs: 0.62 ± 0.55 versus 1.12 ± 0.82, p < 0.05,). Early EPCs from ANFH patients showed impaired migratory capacity (63.8 ± 11.7 versus 152.3 ± 12.4, p < 0.001) and VEGF secretion (50.8 ± 7.2 pg/ml versus 62.8 ± 10.1 pg/ml, p < 0.05). ECFCs from ANFH patients showed decreased tube formation capacity (7.1 ± 2.7 versus 23.8 ± 4.3, p < 0.001) and proliferation.

Discussion

Early EPCs and ECFCs were impaired in number and function in GC-induced ANFH, and their distinct reduced capacity profiles might reflect different roles they played in endothelial dysfunction of GC-induced ANFH.

Introduction

Practically avascular osteonecrosis of the femoral head (ANFH), which represents a devastating condition with a poor prognosis, is a debilitating skeletal disorder affecting especially young patients in their third to fifth decades of life. Avascular osteonecrosis, otherwise known as aseptic necrosis of bone, results from multiple pathological conditions including ischemia, osteocyte apoptosis and necrosis, impaired vascular repair and subsequent loss of structural integrity of the articular surface, thus finally leading to significant clinical morbidity [1]. The most common cause of ANFH is trauma, and the main cause of non-traumatic ANFH is the use of glucocorticoid (GC). Although numerous investigators had postulated several mechanisms involved in ANFH, the exact etiology and pathophysiology of GC-induced non-traumatic ANFH still remains poorly understood.

Recently, growing evidences indicate that microcirculatory disturbance plays a pivotal role during the pathogenesis of GC-induced ANFH [2]. Regional endothelial dysfunction due to continuous exposure to GC in femoral heads results in various disorders such as aberrant vasoactive substances levels, decreased blood flow, endothelial cells apoptosis, microcirculatory thrombus formation, functional microvessels rarefaction, inhibition of angiogenesis and finally osteoblasts and osteocytes death [3]. Therefore, this has prompted investigations on the role of endothelial cells and more importantly, its progenitors – endothelial progenitor cells (EPCs) in GC-induced ANFH.

EPCs initially described by Asahara et al. [4] are now being broadly investigated because of its significant role in vascular homeostasis and angiogenesis. And also it is widely recognized that reduced number and partial dysfunction of EPCs are associated with different disease states and cardiovascular risk [5], [6]. Thus, a number of studies have already implied EPCs as a surrogate biomarker in vascular dysfunction [7]. Our previous study also had found the relationship between EPCs and ANFH [8].

However, recent studies indicated that there was another subtype of EPCs showed distinct morphological phenotype and proliferation pattern from EPCs that Asahara et al. described [9], [10]. The newly so-called endothelial colony forming cells (ECFCs) or late outgrowth endothelial progenitor cells (OECs) demonstrate highly proliferative potential, exhibit self-renewal capacity and are capable of de novo vessel formation in vivo. In contrast, the early EPCs (also known as colony-forming unit endothelial cells or CFU-ECs) expressing hematopoietic markers lack the proliferative capacity characteristic of true progenitor cells, fail to form vascular networks spontaneously and even ingest bacteria, suggesting that they are actually hematopoietic-derived monocytes manifesting some angiogenic properties [11]. And it seems that ECFCs may serve as a potential cell source more effectively for cellular therapies aiming to enhance neovascularization in ischemic area.

Taken together, there is a lack of detailed data concerning the detection and function of ECFCs in ANFH. Given our previous report on reduced number and function of early EPCs in ANFH [8], we questioned whether alterations of ECFCs existed in GC-induced ANFH. Considering significant debate remains about the identity of EPCs, we conducted a study to investigate the levels of early EPCs and ECFCs and compared their functions in GC-induced ANFH patients and age- and sex-matched control subjects.