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

Systemic hypothermia increases PAI-1 expression and accelerates microvascular thrombus formation in endotoxemic mice

INTRODUCTION: Hypothermia during sepsis significantly impairs patient outcome in clinical practice. Severe sepsis is closely linked to activation of the coagulation system, resulting in microthrombosis and subsequent organ failure. Herein, we studied whether systemic hypothermia accelerates microvascular thrombus formation during lipopolysacharide (LPS)-induced endotoxemia in vivo, and characterized the low temperature-induced endothelial and platelet dysfunctions. METHODS: Ferric-chloride induced microvascular thrombus formation was analyzed in cremaster muscles of hypothermic endotoxemic mice. Flow cytometry, ELISA and immunohistochemistry were used to evaluate the effect of hypothermia on endothelial and platelet function. RESULTS: Control animals at 37°C revealed complete occlusion of arterioles and venules after 759 ± 115 s and 744 ± 112 s, respectively. Endotoxemia significantly (p < 0.05) accelerated arteriolar and venular occlusion in 37°C animals (255 ± 35 s and 238 ± 58 s, respectively). This was associated with an increase of circulating endothelial activation markers, agonist-induced platelet reactivity, and endothelial P-selectin and plasminogen activator inhibitor (PAI)-1 expression. Systemic hypothermia of 34°C revealed a slight but not significant reduction of arteriolar (224 ± 35 s) and venular (183 ± 35 s) occlusion times. Cooling of the endotoxemic animals to 31°C core body temperature, however, resulted in a further acceleration of microvascular thrombus formation, in particular in arterioles (127 ± 29 s, p < 0.05 versus 37°C endotoxemic animals). Of interest, hypothermia did not affect endothelial receptor expression and platelet reactivity, but increased endothelial PAI-1 expression and, in particular, soluble PAI-1 antigen (sPAI-Ag) plasma levels. CONCLUSION: LPS-induced endotoxemia accelerates microvascular thrombus formation in vivo, most probably by generalized endothelial activation and increased platelet reactivity. Systemic hypothermia further enhances microthrombosis in endotoxemia. This effect is associated with increased endothelial PAI-1 expression and sPAI-Ag in the systemic circulation rather than further endothelial activation or modulation of platelet reactivity

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

Tissue-Protective Mechanisms of Bioactive Phytochemicals in Flap Surgery

Despite careful preoperative planning, surgical flaps are prone to ischemic tissue damage and ischemia–reperfusion injury. The resulting wound breakdown and flap necrosis increase both treatment costs and patient morbidity. Hence, there is a need for strategies to promote flap survival and prevent ischemia-induced tissue damage. Phytochemicals, defined as non-essential, bioactive, and plant-derived molecules, are attractive candidates for perioperative treatment as they have little to no side effects and are well tolerated by most patients. Furthermore, they have been shown to exert beneficial combinations of pro-angiogenic, anti-inflammatory, anti-oxidant, and anti-apoptotic effects. This review provides an overview of bioactive phytochemicals that have been used to increase flap survival in preclinical animal models and discusses the underlying molecular and cellular mechanisms

Maslinic acid alleviates ischemia/reperfusion-induced inflammation by downregulation of NFκB-mediated adhesion molecule expression

Ischemia/reperfusion (I/R)-induced inflammation is associated with enhanced leukocyte rolling, adhesion and transmigration within the microcirculation. These steps are mediated by hypoxia-triggered signaling pathways, which upregulate adhesion molecule expression on endothelial cells and pericytes. We analyzed whether these cellular events are affected by maslinic acid (MA). Mitochondrial activity and viability of MA-exposed endothelial cells and pericytes were assessed by water-soluble tetrazolium (WST)-1 and lactate dehydrogenase (LDH) assays as well as Annexin V/propidium iodide (PI) stainings. Effects of MA on hypoxia and reoxygenation-induced expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by flow cytometry. The subcellular localization of the NFκB subunit p65 was analyzed by immunofluorescence and Western blot. I/R-induced leukocytic inflammation was studied in MA- and vehicle-treated mouse dorsal skinfold chambers by intravital fluorescence microscopy and immunohistochemistry. MA did not affect viability, but suppressed the mitochondrial activity of endothelial cells. Furthermore, MA reduced adhesion molecule expression on endothelial cells and pericytes due to an inhibitory action on NFκB signaling. Numbers of adherent and transmigrated leukocytes were lower in post-ischemic tissue of MA-treated mice when compared to vehicle-treated controls. In addition, MA affected reactive oxygen species (ROS) formation, resulting in a diminished oxidative DNA damage. Hence, MA represents an attractive compound for the establishment of novel therapeutic approaches against I/R-induced inflammation

Protein Kinase CK2—A Putative Target for the Therapy of Diabetes Mellitus?

Since diabetes is a global epidemic, the development of novel therapeutic strategies for the treatment of this disease is of major clinical interest. Diabetes is differentiated in two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM arises from an autoimmune destruction of insulin-producing β-cells whereas T2DM is characterized by an insulin resistance, an impaired insulin reaction of the target cells, and/or dysregulated insulin secretion. In the past, a growing number of studies have reported on the important role of the protein kinase CK2 in the regulation of the survival and endocrine function of pancreatic β-cells. In fact, inhibition of CK2 is capable of reducing cytokine-induced loss of β-cells and increases insulin expression as well as secretion by various pathways that are regulated by reversible phosphorylation of proteins. Moreover, CK2 inhibition modulates pathways that are involved in the development of diabetes and prevents signal transduction, leading to late complications such as diabetic retinopathy. Hence, targeting CK2 may represent a novel therapeutic strategy for the treatment of diabetes

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

Targeting sphingosine kinase-1 with the low MW inhibitor SKI-5C suppresses the development of endometriotic lesions in mice

Background and Purpose Limited evidence suggests that the sphingosine-1-phosphate/sphingosine kinase 1 (S1P/SPHK1) signalling pathway is involved in the pathogenesis of endometriosis. Therefore, we analyzed in this study whether the inhibition of SPHK1 and, consequently, decreased levels of S1P affected the vascularization and growth of endometriotic lesions. Experimental Approach Endometriotic lesions were surgically induced in the peritoneal cavity and the dorsal skinfold chamber of female BALB/c mice. The animals received a daily dose of the SPHK1 inhibitor SKI-5C or vehicle (control). Analyses involved the determination of lesion growth, cyst formation, microvessel density and cell proliferation within peritoneal endometriotic lesions by means of high-resolution ultrasound imaging, caliper measurement, histology and immunohistochemistry. In the dorsal skinfold chamber model the development of newly formed microvascular networks and their microhemodynamic parameters within endometriotic lesions were investigated by means of intravital fluorescence microscopy. Key Results SKI-5C significantly inhibited the development and vascularization of peritoneal endometriotic lesions, as indicated by a reduced growth and cyst formation, a lower microvessel density and a suppressed cell proliferation, when compared to vehicle-treated controls. Endometriotic lesions in dorsal skinfold chambers of SKI-5C-treated animals exhibited a significantly smaller lesion size, lower functional microvessel density, smaller microvessel diameters and a reduced blood perfusion of the newly developing microvascular networks. Conclusions and Implications SPHK1/S1P signalling promotes the establishment and progression of endometriotic lesions. The inhibition of this pathway suppresses the development of endometriotic lesions, suggesting SPHK1 as a potential novel target for future endometriosis therapy

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

Bromelain Protects Critically Perfused Musculocutaneous Flap Tissue from Necrosis

Bromelain has previously been shown to prevent ischemia-induced necrosis in different types of tissues. In the present study, we, therefore, evaluated for the first time, the tissue-protective effects of bromelain in musculocutaneous flaps in mice. Adult C57BL/6N mice were randomly assigned to a bromelain treatment group and a control group. The animals were treated daily with intraperitoneal injections of 20 mg/kg bromelain or saline (control), starting 1 h before the flap elevation throughout a 10-day observation period. The random-pattern musculocutaneous flaps were raised on the backs of the animals and mounted into a dorsal skinfold chamber. Angiogenesis, nutritive blood perfusion and flap necrosis were quantitatively analyzed by means of repeated intravital fluorescence microscopy over 10 days after surgery. After the last microscopy, the flaps were harvested for additional histological and immunohistochemical analyses. Bromelain reduced necrosis of the critically perfused flap tissue by ~25%. The bromelain-treated flaps also exhibited a significantly higher functional microvessel density and an elevated formation of newly devel oped microvessels in the transition zone between the vital and necrotic tissues when compared to the controls. Immunohistochemical analyses demonstrated a markedly lower invasion of the myeloperoxidase-positive neutrophilic granulocytes and a significantly reduced number of cleaved caspase 3-positive apoptotic cells in the transition zone of bromelain-treated musculocutaneous flaps. These findings indicate that bromelain prevents flap necrosis by maintaining nutritive tissue perfusion and by suppressing ischemia-induced inflammation and apoptosis. Hence, bromelain may represent a promising compound to prevent ischemia-induced flap necrosis in clinical practice