Silvan Klein

Angiogenesis: The role of PDGF-BB on Adiopse-tissue derived Stem Cells (ASCs)

Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASCs migration towards PDGF-BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated with proliferation, anti-apoptosis or differentiation.

In vitro studies investigating the effect of subcutaneous phosphatidylcholine injections in the 3T3-L1 adipocyte model: lipolysis or lipid dissolution?

Background: The demand for lipolytic injection therapies for aesthetic indications increases continuously. The substance most frequently discussed in this context is phosphatidylcholine solubilized in sodium deoxycholate, a composition known in Europe as Lipostabil (Artesan Pharma, Lüchow, Germany). The evidence for its lipolytic effect is based mainly on clinical studies that suggest a decrease in adipose tissue volume after subcutaneous injections. However, neither the actual effect nor the effective supplemental effect of Lipostabil has been clearly identified so far. The purpose of this study was to investigate the effects caused by lipolytic formulations on adipocytes using an in vitro model. Methods: 3T3-L1 adipocytes in 12-well plates were exposed to varying doses of isolated phosphatidylcholine, deoxycholate, and the combination of both (Lipostabil). Subsequently, changes in the cell membrane integrity were evaluated microscopically, and assays measuring the amount of glycerol as a biochemical lipolysis substrate (lipolysis assay) together with dimethyl thiazolyl diphenyl tetrazolium assays were performed to quantify the lipolytic effect and the cell viability. Results: Deoxycholate reduced cell viability significantly (p < 0.05), even at low concentrations. Neither phosphatidylcholine nor deoxycholate led to a significant (p < 0.05) induction of a lipolytic pathway. Lipostabil, the combination of deoxycholate and phosphatidylcholine, led to a significant (p < 0.05) decrease in cell viability at low doses and to a highly significant (p < 0.01) reduction at high doses. The loss in cell viability is attributable to changes in the cell membrane integrity. Conclusions: These results suggest that no enzymatic lipolytic pathway is induced. The decrease in volume after Lipostabil injections is likely attributable to the detergent effect of deoxycholate.

Evidence‐based topical management of chronic wounds according to the T.I.M.E. principle

The number of patients suffering from chronic wound healing disorders in Germany alone is estimated to be 2.5–4 million. Therapy related expenses reach 5–8 billion Euros annually. This number is partially caused by costly dressing changes due to non‐standardized approaches and the application of non‐evidence‐based topical wound therapies. The purpose of this paper is to elucidate a straightforward principle for the management of chronic wounds, and to review the available evidence for the particular therapy options. The T.I.M.E.‐principle (Tissue management, Inflammation and infection control, Moisture balance, Epithelial [edge] advancement) was chosen as a systematic strategy for wound bed preparation. Literature was retrieved from the PubMed and Cochrane Library databases and subjected to selective analysis.

Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

Early magnetic resonance imaging in spinal cord injury without radiological abnormality in adults: a retrospective study.

BACKGROUND The purpose of this study was to describe the clinical and imaging characteristics of patients experiencing blunt spinal trauma without radiological abnormalities but transient or persistent neurological deficits. METHODS This retrospective study analyzed plain radiographs, computed tomographic scans, and magnetic resonance images of patients with spinal cord injury without radiological abnormality (SCIWORA) who were admitted to a Level I trauma center. Neurologic status, Frankel grade, and short-term patient outcome were assessed. RESULTS Of 1,604 patients experiencing blunt spinal trauma, 21 (12 men and 9 women) with a mean age of 35.5 years (range, 16.2–70.9 years) presented with a clinicoradiographic mismatch. Magnetic resonance imaging (MRI) was available in 15 patients. In seven patients (46.6%), MRI revealed either neural (n = 2, 13.3%) or extraneural (n = 5, 33.3%) spinal abnormalities. Importantly, in eight patients (53.3%), no spinal abnormalities were visible on MRI. Furthermore, subgroup analysis revealed no prognostic value regarding the presence or absence of detectable spinal injuries. CONCLUSION Spinal abnormalities were not detected on MRI in a substantial proportion of patients presenting with SCIWORA. The prognostic value of MRI findings in SCIWORA needs to be validated by future studies. LEVEL OF EVIDENCE Epidemiological study, level V.

Limitation of in vivo models investigating angiogenesis in breast cancer

MSCs reside within their niche and pathologic conditions such as hypoxia and inflammation can lead to mobilization and migration of Mesenchymal Stem Cells (MSCs). Xenograft animal models using immundeficient mouse demonstrated that MSCs migrated to and distributed throughout the tumors and were found to engraft into tumor stroma and vasculature. In contrast, MSCs primarily incorporated within tumor-capsula and did not invade the tumor using immuncompetent tumor allograft models. Here we hypothesize that MSCs migrate primarily towards an inflammatory milieu independent of the underlying biological process causing the inflammation. Murine MSCs (mASCs) were isolated from subcutaneous fat tissues and transduced at passage 0 with lentiviral vector encoding green fluorescent protein (GFP) and luciferase reporter. Breast cancer was established in BALB/c mice by subcutaneous injection of 4T1 cells into the left mammary fat pad. E. coli were injected subcutaneously in the right 4th mammary fat pad. After 24 h luciferase labeled mASCs were administered intraperitoneal (i.p.) and monitored with IVIS Bioluminescence camera for 72 hours. Control group received either tumor implantation or E. coli injection. MSCs significantly migrated towards tumor when compared to control mice without tumor or inflammatory process. However, mASCs injected in 4T1 bearing mice with E. coli only migrated towards the bacterial inflammatory focus. Our results substantiate the notation the MSCs response predominantly to the inflammatory milieu created by bacteria or tumor rather than specifically to the tumor. Thus, it is suggested that the migration of MSCs in immunodeficient mice depends on cancer secreted cytokines due to the lack of the inflammatory response by the immune system. Therefore, in vivo studies investigating the role of MSCs in tumor angiogenesis have shown controversy results and should be interpreted with caution in terms of tumor secreted cytokine dependent stem cell migration.

Contrast enhanced ultrasound (CEUS) – an unique monitoring technique to assess microvascularization after buried flap transplantation

OBJECTIVE Incidence of patients requiring complex soft tissue or osseous reconstruction has dramatically increased. However most of the monitoring systems have limitations in tissue penetration and are not able to detect microvascular complications after transplantation of so-called buried-flaps, that have no contact to the surface.Aim of the study was to assess contrast enhanced ultrasound (CEUS) as monitoring tool after buried flap transplantations. METHODS 20 patients were examined after buried flap transplantation using CEUS. Quantitative perfusion analysis (TIC) was performed with an integrated perfusion software using stored cine-loops. Two perfusion-parameters, time to PEAK (TtoPk) and area under the curve (Area), were evaluated using TIC analysis. RESULTS Minor complications were observed in 3 patients. In these patients a delayed contrast agent wash-in and wash-out was observed. Additionally the perfusion values TtoPk (sec.) and Area (relative Units) were clearly different in the patients with minor complications: TtoPk: 32.0 sec; Area 425.5 rU (without complication), TtoPk: 38.6 sec.; Area: 18.3 rU (wound healing disturbance) and TtoPk: 14.4 sec.; Area: 105.9 rU (hematoma). CONCLUSION As CEUS can assess microvascularization almost depth-independent, CEUS is an unique method to assess global flap perfusion after buried flap transplantation.

Transcription Profile in Sporadic Multiple Symmetric Lipomatosis Reveals Differential Expression at the Level of Adipose Tissue-Derived Stem Cells.

Background: The cause of the rare fat distribution disorder multiple symmetric lipomatosis is unknown. Independent reports suggest a higher proliferative activity, hormone resistance, and involvement of mitochondrial function in the disease. Methods: The authors performed morphologic comparison of affected and unaffected tissues in five unrelated patients and generated adipose-derived stem cell cultures from the tissue samples and characterized them as a possible cellular model of multiple symmetric lipomatosis evolution. The authors investigated proliferative activity and the expression of genes relevant to disease processes. Results: There was no difference in the morphologic appearance and the surface marker profile. Stem cells from lipomatous tissue showed significantly higher proliferative activity. Polymerase chain reaction arrays showed marked changes in genes associated with proliferation, hormonal regulation, and mitochondria. The authors show that multiple symmetric lipomatosis tissue is morphologically and histologically different from regular subcutaneous fat. Conclusions: This study indicates an involvement of mesenchymal stem cells in the pathogenesis of multiple symmetric lipomatosis and that the evolution of multiple symmetric lipomatosis tissue is a process driven by an inherent defect of the respective cell clone(s). Further molecular genetics and functional analysis will be required to unravel the pathogenetic mechanism underlying the derailment in fat cell metabolism and proliferation. Here, the authors show for the first time that adipose-derived stem cells exhibit many characteristics previously described for native multiple symmetric lipomatosis fat tissue and propose that they are therefore an excellent tool for further functional investigations in multiple symmetric lipomatosis and other disorders of the fat tissue. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, V.

Hypoxic downregulation of cellular proliferation and loss of phenotype stability in human osteoblasts is mediated by HIF-1α

Both, skeletal development and fracture healing depend on an orchestrated sequence of cellular growth and differentiation processes. Regional changes in tissue oxygen tension were proposed as key regulators of osteoblast proliferation and phenotype. Hypoxia results in the stabilization of hypoxia-inducible factor-1α (HIF-1α), thus influencing expression of a multitude of genes required for cellular adaptation. In the present study we dissected the effects of HIF-1α on cellular proliferation and gene expression of primary human osteoblasts. Primary human osteoblasts were studied by transfecting siRNA and plasmids coding for human HIF-1α. Gene expression was analyzed by western blot and quantitative PCR. Functional assays were performed to study HIF-1α function, i.e. proliferation and cell cycle analysis. As previously reported exposure to hypoxia led to a stabilization of HIF-1α on protein level and resulted in reduced rates of proliferation and osteocalcin expression. Furthermore, the expression of the proproliferative gene survivin was significantly reduced (p < 0.01). Knock down of HIF-1α attenuated hypoxic downregulation of proliferation (p < 0.05), and osteocalcin (p < 0.05) as well as survivin (p < 0.05) expression significantly. Importantly, the isolated overexpression of HIF-1α impaired proliferative activity and led to significantly reduced rates of expression of osteocalcin (p < 0.05) and survivin (p < 0.01). The present study shows that HIF-1α might reduce proliferation and survivin expression in primary human osteoblasts independently from cellular hypoxia. Furthermore, HIF-1α promoted the loss of the characteristic osteoblastic marker, osteocalcin in vitro. These findings underline the important role of HIF-1α in bone physiology and pathophysiology. Modulating HIF-1α function in hypoxic environments could be of value for future therapeutic approaches.

Vascular malformations of upper and lower extremity - from radiological interventional therapy to surgical soft tissue reconstruction – an interdisciplinary treatment

This article presents our experience in managing peripheral vascular malformations of upper and lower extremities over a 4-year period in a series of 46 patients of the Department of Plastic Surgery treated in the Interdisciplinary Center of Vascular Anomalies (ICVA) at the University of Regensburg. The patients presented vascular malformations of upper and lower extremity and were selected from our prospective vascular anomalies file archive from 2012 to 2016. During this period in the ICVA at University of Regensburg were performed more than 1400 radiological interventional treatments in patients with vascular malformations.The purpose of this retrospective study was to review combined embolotherapy, sclerotherapy (embolo/sclerotherapy), and surgical procedures (surgical excision and soft tissue reconstruction) to manage vascular malformations. Treatments were principally induced to reduce pain, daily physical limitations, social discomfort and recover tegument continuity after ulceration.The 46 patients were first examined with noninvasive radiological procedures. After diagnosis was posed, embolo/sclerotherapy, surgical procedures and clinically as well as radiological follow-ups were coordinated and established by the multidisciplinary team. All vascular malformations were categorized according to the classification approved at the April 2014 General Assembly of International Society for the Study of Vascular Anomalies (ISSVA) in Melbourne, Australia. Arteriovenous malformations (AVMs) were further classified following the Cho-Do and Schobinger classification.Embolo/sclerotherapy shows to be the most appropriate procedure in vascular malformations treatment. Nevertheless was found that in case of complications or lack of improvement as well as to improve functional or aesthetical results, a following partial or complete surgical excision and immediate soft tissue reconstruction seems to be the gold-standard treatment. In addition, the precise clinical and radiological diagnosis as well as an intensive postoperative patient care have a significant positive influence on the clinical outcome and patient satisfaction while decreasing morbidity and recurrence during early and late follow-up.Vascular malformations require a multidisciplinary approach and individual treatment after complex excision and indispensable reconstruction.