Kristoff Verdonck

Integration of Blood Outgrowth Endothelial Cells in Dermal Fibroblast Sheets Promotes Full Thickness Wound Healing

Vascularization is the cornerstone of wound healing. We introduced human blood outgrowth endothelial cells (hBOEC) in a self‐assembled human dermal fibroblast sheet (hDFS), intended as a tissue‐engineered dermal substitute with inherent vascular potential. hBOEC were functionally and molecularly different from early endothelial progenitor cells and human umbilical vein endothelial cells (HUVEC). hBOEC alone, unlike HUVEC, efficiently revascularized and re‐oxygenated the wound bed, both by active incorporation into new vessels and by trophic stimulation of host angiogenesis in a dose‐dependent manner. Furthermore, hBOEC alone, but not HUVEC, accelerated epithelial coverage and matrix organization of the wound bed. In addition, integration of hBOEC in hDFS not only further improved vascularization, epithelial coverage and matrix organization but also prevented excessive wound contraction. In vitro analyses with hBOEC, fibroblasts and keratinocytes revealed that these effects were both due to growth factor crosstalk and to short cutting hypoxia. Among multiple growth factors secreted by hBOEC, placental growth factor mediated at least in part the beneficial effects on keratinocyte migration and proliferation. Overall, this combined tissue engineering approach paves the way for clinical development of a fully autologous vascularized dermal substitute for patients with large skin defects that do not heal properly. STEM CELLS 2010;28:1165–1177

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

Aims/hypothesisAs current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function.MethodsHuman BOEC, 5 × 105, were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated.ResultsAlthough vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient.Conclusions/interpretationThe present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved

Background Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age‐matched (ACON) and healthy young (CON) controls. Methods and Results We isolated 3.6±0.6 BOEC colonies/100×106 mononuclear cells (MNCs) from 60‐mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×106 MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×106 MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2+/CD31+/CD146+) and progenitor (CD34+/CD133−) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three‐dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin‐2 (1.4±0.3×105 pg/106 ICMP‐BOECs) and placental growth factor (5.8±1.5×103 pg/106 ICMP BOECs), independent of age or ischemic disease. Senescence‐associated β‐galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High‐resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831). Conclusions BOECs can be successfully culture‐expanded from patients with ICMP. In contrast to impaired functionality of ICMP‐derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease.

Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes

We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5 x 105 BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.

Cell and Gene Transfer Strategies for Vascularization During Skin Wound Healing

Adequate vascularization is pivotal to skin wound healing. Therefore, designing efficient revascularization strategies based on the mechanisms behind electromechanical stimulation of wound vascularization would be beneficial to the growing number of patients in need of improved wound healing. Recent attention has centered on applying gene/protein transfer and cell differentiation/transplantation approaches to stimulate and mimic the molecular events occurring during wound revascularization. Although both gene/protein transfer and cell differentiation/transplantation are faced with important challenges, researchers have made tremendous advances and shown both strategies to be a promising approach. In this chapter, we give an overview of the myriad of molecular players involved in neovascularization. We also discuss the molecular mechanisms of neovascularization during wound healing and provide an in-depth review on neovascular strategies and techniques for wound healing and tissue-engineered skin equivalents.

LOP25: Angiogenic Factors Placental Growth Factor (PlGF) and Vascular Endothelial Growth Factor (VEGF) are in Part Responsible for the Beneficial Effects of Human Blood Outgrowth Endothelial Cells (hBOECs) and Dermal Fibroblast Sheets (hDFS) on Wound Healing

28d. Gluteal muscles were harvested and processed for histology. The area of inflammation surrounding each suture was quantified and compared between groups. RESULTS: PEA-coated silk sutures resulted in significantly decreased mean areas of inflammation compared with non-coated controls after 7 and 28d (686,897μm2±99,646μm2 v. 2,095,447μm2±385,461μm2, p2 v. 272,230μm2±40,156μm2, p2 v. 2,502,000μm2±462,461μm2, p CONCLUSION: PEA-coating significantly decreases the local immune response to inflammatory plain-gut and silk sutures. Although further study is warranted prior to clinical use, suture modification via PEAcoating offers a promising means to improve suture biocompatibility and minimize surgical site morbidity.