Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.

Proteomic analysis of porcine bone-conditioned medium.

PURPOSE Autografts are considered to support bone regeneration. Paracrine factors released from cortical bone might contribute to the overall process of graft consolidation. The aim of this study was to characterize the paracrine factors by means of proteomic analysis. MATERIALS AND METHODS Bone-conditioned medium (BCM) was prepared from fresh bone chips of porcine mandibles and subjected to proteomic analysis. Proteins were categorized and clustered using the bioinformatic tools UNIPROT and PANTHER, respectively. RESULTS Proteomic analysis showed that BCM contains more than 150 proteins, of which 43 were categorized into "secreted" and "extracellular matrix." Growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration, eg, pleiotrophin, galectin-1, transforming growth factor beta (TGF-β)-induced gene (TGFBI), lactotransferrin, insulin-like growth factor (IGF)-binding protein 5, latency-associated peptide forming a complex with TGF-β1, and TGF-β2, were discovered. CONCLUSION The present results demonstrate that cortical bone chips release a large spectrum of proteins with the possibility of modulating cellular aspects of bone regeneration. The data provide the basis for future studies to understand how these paracrine factors may contribute to the complex process of graft consolidation.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Current developments in the use of stem cell for therapeutic neovascularisation: is the future therapy "cell-free"?

The plasticity and self-regenerative properties of stem cells have opened new avenues in regenerative medicine. Greater understanding of the biology of stem cells is followed by growing expectations of a rapid translation into alternative therapeutic options. Recent preclinical studies and clinical trials employing stem and progenitor cells from different sources have shown encouraging results. However, their underlying mechanisms are still poorly understood, the potential adverse effects and the discrepancy in efficacy remain to be further investigated. Their essential role in vessel regeneration has made endothelial progenitor cells (EPC) a suitable candidate for therapeutic applications aiming at tissue revascularisation. Recent evidence suggests that EPC contribute to neovascularisation not only by direct participation in tissue homeostasis but mainly via paracrine mechanisms. In future, novel therapeutic strategies could be based on EPC paracrine factors or synthetic factors, and replace cell transplantation.

Biology of Bone Metastases in Prostate Cancer

Advanced-stage prostate cancer (PCa) patients are often diagnosed with bone metastases. Bone metastases remain incurable and therapies are palliative. PCa cells prevalently cause osteoblastic lesions, characterized by an excess of bone formation. The prevailing concept indicates that PCa cancer cell secrete an excess of paracrine factors stimulating osteoblasts directly or indirectly, thereby leading to an excess of bone formation. The exact mechanisms by which bone formation stimulates PCa cell growth are mostly elusive. In this review, the mechanisms of PCa cancer cell osteotropism, the cancer cell-induced response within the bone marrow/bone stroma, and therapeutic stromal targets will be summarized.