Evaluation of VEGF‐mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast‐mediated crosstalk to endothelial cells

Abstract

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing

“…To examine if sub-chondral osteoblasts near the vascularisation area expressed different levels of Sulf1 and Sulf2 or their shorter variants that could promote angiogenesis, we compared these cultures with cancellous bone derived osteoblasts in the presence and absence of PGE2 known to promote VEGF expression by such cells (Clarkin et al 2008). The osteoblast cultures prepared from both cancellous and subchondral bone as representatives of in vivo endochondral ossification also demonstrated generally similar changes in Sulf1 and Sulf2 except with a slightly higher level expression of Sulf2 relative to Sulf1, indicating similar roles of Sulf1 and Sulf2 during in vitro differentiation of osteoblasts.…”

“…Human skeletal tissues: Primary human osteoblast cell cultures were prepared from bone segments taken during routine shoulder operations at the Hospital of St. John and St. Elizabeth, London following patient consent before each operation as previously described (Clarkin et al 2008). Samples of human articular cartilage from different age groups were also obtained following patient consent before each operation within 6 h of surgery from weight-bearing regions of femoral condyles from patients undergoing amputation or joint replacement for osteosarcoma or chondrosarcoma distant from the joint, and in all cases, joints were not involved in the tumor pathology as previously described (Hickery et al 2003).…”

“…To determine if the levels or nature of Sulf1 or Sulf2 variants varied in cancellous and sub-chondral bone-derived osteoblasts, primers specific for each Sulf variant mRNA were used for RT PCR analysis of prostaglandin E2 (PGE2) treated cancellous and sub-chondral bone-derived osteoblasts since the full length SULFs are known to inhibit VEGF activity and PGE2 treatment has been shown to induce VEGF production (Clarkin et al 2008). Both Sulf1 and Sulf2 variants were expressed by all osteoblasts and PGE2 treatment produced no effect on their relative expression levels examined by this procedure (Figure 3).…”

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

“…These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts [1,2] . BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites.…”

In vitro models for the evaluation of angiogenic potential in bone engineering

“…VEGF-stimulated ECs release prostaglandins that strongly promote VEGF release by osteoblasts [35] . ECs co-cultured with OBs show an increased production of collagen type I [36] .…”