Macrophages promote network formation and maturation of transplanted adipose tissue-derived microvascular fragments

Adipose tissue-derived microvascular fragments rapidly reassemble into microvascular networks within implanted scaffolds. Herein, we analyzed the contribution of macrophages to this process. C57BL/6 mice received clodronate (clo)-containing liposomes for macrophage depletion, whereas animals treated with phosphate-buffered-saline-containing liposomes served as controls. Microvascular fragments were isolated from clo- and phosphate-buffered-saline-treated donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of clo- and phosphate-buffered-saline-treated recipient mice. The implants' vascularization and incorporation were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. Compared to controls, matrices within clo-treated animals exhibited a significantly reduced functional microvessel density. Moreover, they contained a lower fraction of microvessels with an α-smooth muscle actin (SMA)+ cell layer, indicating impaired vessel maturation. This was associated with a deteriorated implant incorporation. These findings demonstrate that macrophages not only promote the reassembly of microvascular fragments into microvascular networks, but also improve their maturation during this process

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

Aims The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. Methods Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. Results We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. Conclusion These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation

Darbepoetin-α increases the blood volume flow in transplanted pancreatic islets in mice

Aims The minimal-invasive transplantation of pancreatic islets is a promising approach to treat diabetes mellitus type 1. However, islet transplantation is still hampered by the insufficient process of graft revascularization, leading to a poor clinical outcome. Accordingly, the identification of novel compounds, which accelerate and improve the revascularization of transplanted islets, is of great clinical interest. Previous studies have shown that darbepoetin (DPO)-α, a long lasting analogue of erythropoietin, is capable of promoting angiogenesis. Hence, we investigated in this study whether DPO improves the revascularization of transplanted islets. Methods Islets were isolated from green fluorescent protein-positive FVB/N donor mice and transplanted into dorsal skinfold chambers of FVB/N wild-type animals, which were treated with DPO low dose (2.5 µg/kg), DPO high dose (10 µg/kg) or vehicle (control). The revascularization was assessed by repetitive intravital fluorescence microscopy over an observation period of 14 days. Subsequently, the cellular composition of the grafts was analyzed by immunohistochemistry. Results The present study shows that neither low- nor high-dose DPO treatment accelerates the revascularization of free pancreatic islet grafts. However, high-dose DPO treatment increased the blood volume flow of the transplanted islet. Conclusions These findings demonstrated that DPO treatment does not affect the revascularization of transplanted islets. However, the glycoprotein increases the blood volume flow of the grafts, which results in an improved microvascular function and may facilitate successful transplantation

The regulatory mechanisms of NG2/CSPG4 expression

Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration and angiogenesis. NG2 is frequently used as a marker for the identification and characterization of certain cell types, but little is known about the mechanisms regulating its expression. In this review, we provide evidence that the regulation of NG2 expression underlies inflammation and hypoxia and is mediated by methyltransferases, transcription factors, including Sp1, paired box (Pax) 3 and Egr-1, and the microRNA miR129-2. These regulatory factors crucially determine NG2-mediated cellular processes such as glial scar formation in the central nervous system (CNS) or tumor growth and metastasis. Therefore, they are potential targets for the establishment of novel NG2-based therapeutic strategies in the treatment of CNS injuries, cancer and other conditions of these types

Parathyroid hormone stimulates bone regeneration in an atrophic non-union model in aged mice

Background Non-union formation still represents a major burden in trauma and orthopedic surgery. Moreover, aged patients are at an increased risk for bone healing failure. Parathyroid hormone (PTH) has been shown to accelerate fracture healing in young adult animals. However, there is no information whether PTH also stimulates bone regeneration in atrophic non-unions in the aged. Therefore, the aim of the present study was to analyze the efect of PTH on bone regeneration in an atrophic non-union model in aged CD-1 mice. Methods After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fxation. The animals were treated daily with either 200 mg/kg body weight PTH 1–34 (n=17) or saline (control; n=17) subcutaneously. Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses. Results In PTH-treated animals bone formation was markedly improved when compared to controls. This was associated with an increased bending stifness as well as a higher number of tartrate-resistant acid phosphatase (TRAP)- positive osteoclasts and CD31-positive microvessels within the callus tissue. Furthermore, PTH-treated aged animals showed a decreased infammatory response, characterized by a lower number of MPO-positive granulocytes and CD68-positive macrophages within the bone defects when compared to controls. Additional Western blot analyses demonstrated a signifcantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K) in PTH-treated mice. Conclusion Taken together, these fndings indicate that PTH is an efective pharmacological compound for the treatment of non-union formation in aged animals

Parathyroid hormone [1-34] improves articular cartilage surface architecture and integration and subchondral bone reconstitution in osteochondral defects in vivo

SummaryObjectiveThe 1-34 amino acid segment of the parathyroid hormone (PTH [1-34]) mediates anabolic effects in chondrocytes and osteocytes. The aim of this study was to investigate whether systemic application of PTH [1-34] improves the repair of non-osteoarthritic, focal osteochondral defects in vivo.DesignStandardized cylindrical osteochondral defects were bilaterally created in the femoral trochlea of rabbits (n = 8). Daily subcutaneous injections of 10 μg PTH [1-34]/kg were given to the treatment group (n = 4) for 6 weeks, controls (n = 4) received saline. Articular cartilage repair was evaluated by macroscopic, biochemical, histological and immunohistochemical analyses. Reconstitution of the subchondral bone was assessed by micro-computed tomography. Effects of PTH [1-34] on synovial membrane, apoptosis, and expression of the PTH receptor (PTH1R) were determined.ResultsSystemic PTH [1-34] increased PTH1R expression on both, chondrocytes and osteocytes within the repair tissue. PTH [1-34] ameliorated the macro- and microscopic aspect of the cartilaginous repair tissue. It also enhanced the thickness of the subchondral bone plate and the microarchitecture of the subarticular spongiosa within the defects. No significant correlations were established between these coexistent processes. Apoptotic levels, synovial membrane, biochemical composition of the repair tissue, and type-I/II collagen immunoreactivity remained unaffected.ConclusionsPTH [1-34] emerges as a promising agent in the treatment of focal osteochondral defects as its systemic administration simultaneously stimulates articular cartilage and subchondral bone repair. Importantly, both time-dependent mechanisms of repair did not correlate significantly at this early time point and need to be followed over prolonged observation periods

Erythropoietin accelerates the revascularization of transplanted pancreatic islets

Background and Purpose Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets. Experimental Approach Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kg−1) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6‐day period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocin‐induced diabetic mouse model was used to study the effect of EPO‐pretreatment on the endocrine function of the grafts. Key Results EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPO‐pretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPO‐pretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation. Conclusion and Implications These findings demonstrate that, particularly, short‐term EPO‐pretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events

Cilostazol promotes blood vessel formation and bone regeneration in a murine non-union model

Non-unions represent a major complication in trauma and orthopedic surgery. Many factors contribute to bone regeneration, out of which an adequate vascularization has been recognized as crucial. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in a variety of preclinical studies. Hence, we herein investigated the effects of cilostazol on bone regeneration in an atrophic non-union model in mice. For this purpose, a 1.8 mm femoral segmental defect was stabilized by pin-clip fixation and the animals were treated daily with 30 mg/kg body weight cilostazol or saline (control) per os. At 2, 5 and 10 weeks after surgery the healing of femora was analyzed by X-ray, biomechanics, photoacoustic imaging, and micro-computed tomography (µCT). To investigate the cellular composition and the growth factor expression of the callus tissue additional histological, immunohistochemical and Western blot analyses were performed. Cilostazol-treated animals showed increased bone formation within the callus, resulting in an enhanced bending stiffness when compared to controls. This was associated with a more pronounced expression of vascular endothelial growth factor (VEGF), a higher number of CD31-positive microvessels and an increased oxygen saturation within the callus tissue. Furthermore, cilostazol induced higher numbers of tartrate-resistant acidic phosphate (TRAP)-positive osteoclasts and CD68-positive macrophages. Taken together, these findings demonstrate that cilostazol is a promising drug candidate for the adjuvant treatment of atrophic non-unions in clinical practice

Complete shutdown of microvascular perfusion upon hepatic cryothermia is critically dependent on local tissue temperature

Since microvascular dysfunction with complete circulatory arrest and, thus, prolongation of tissue ischaemia is considered a potential mechanism for cell necrosis following hepatic cryosurgery, we determined the temperature necessary for induction of complete nutritive perfusion failure in cryothermia-treated rat livers. After localization of the cryoprobe with seven thermocouples and application of a single or double freeze–thaw cycle, in vivo fluorescence microscopy of the cryoinjured left lobe was performed over a 2-h period using a computer-controlled stepping motor, which guaranteed analysis of the identical liver tissue segments with exact allocation of the thermocouples and thus determination of tissue temperature. Cryothermia resulted in a central non-perfused part of injury, surrounded by a heterogeneously perfused peripheral zone. The non-perfused area after single and double freezing continuously increased over the first 90-min period due to a successive shutdown of perfusion within the peripheral border zone. Analysis of the thermocouples' temperature at the end of freezing revealed the 0°C-front at 11.7 mm (single freeze–thaw cycle) and 12.1 mm (double freeze–thaw cycle) distant from the centre of the cryoprobe, which exactly corresponds with the initial (30 min) expansion of the area with nutritive perfusion failure. The increased non-perfused tissue area at 2 h conformed a critical border temperature between 8.29 ± 1.63°C and 9.07 ± 0.24°C. From these findings, we conclude that freezing of liver tissue to temperatures of at least < 0°C causes complete/irreversible perfusion failure, which consequently will result in cell death and tissue necrosis, and may thus be supposed as a prerequisite for the safe and successful application of cryosurgery in hepatic tumour ablation. © 2000 Cancer Research Campaig