Filip Simunovic

miR-126 regulates platelet-derived growth factor receptor-α expression and migration of primary human osteoblasts

Abstract Adequate vascularization is an essential requirement for bone development, fracture healing and bone tissue engineering. We have previously described the coculture of primary human osteoblasts (hOBs) and human endothelial cells (HUVECs), designed to investigate the interactions between these cells. In this system, we showed that cocultivation of these two cell types leads to a downregulation of platelet-derived growth factor receptor-α (PDGFR-α) in hOBs, which was a consequence of reduced mRNA stability. In the current study we investigated the possible involvement of microRNAs in this process. Firstly, we performed a microarray analysis of osteoblastic miRNAs following cocultivation with HUVECs, revealing an upregulation of miR-126. This result was confirmed by RT-qPCR, and we observed that the increase is dependent on direct cell-to-cell contacts. Gain-of-function and loss-of-function experiments showed that miR-126 is a negative regulator of PDGFR-α mRNA. Additionally, migration of hOBs was inhibited by miR-126 overexpression and stimulated by miR-126 inhibition. Addition of PDGFR-α blocking antibody to hOB culture also inhibited hOB migration. There was no effect of miR-126 modulation on osteoblast proliferation, apoptosis rate or differentiation. In conclusion, we report that the miR-126/PDGFR-α system regulates the migratory behavior of human osteoblasts, without exerting effects on cell survival and differentiation.

Aesthetic surgery performed by plastic surgery residents: an analysis of safety and patient satisfaction.

BackgroundAesthetic surgery is an integral component of plastic surgery. Despite its importance, adequate training in aesthetic surgery is met with challenges. Although the educational benefit of resident clinics has been demonstrated, such clinics are rarely found outside the United States. The objective of the present study was to assess safety and patient satisfaction associated with aesthetic surgery procedures performed by plastic surgery residents at a German academic medical center. MethodsThe study had 2 components, namely, a retrospective chart review and an administration of a patient satisfaction survey. Only patients who underwent a surgical intervention by a plastic surgery resident between 2003 and 2011 were included in the study. Parameters of interest included age, sex, procedure performed, number of procedures, revenue (in &OV0556;), length of follow-up, revision rate, and postoperative complication rate. Patient satisfaction was assessed by the client satisfaction questionnaire-8. ResultsA total of 273 aesthetic procedures were performed in 206 patients with an increase in recent years. The median follow-up period was 49.5 months. The most frequently performed procedures were liposuction (n = 59), breast augmentation (n = 53), and upper eyelid blepharoplasty (n = 31). One hundred ninety-two (90.3%) patients had an uneventful postoperative course. The client satisfaction questionnaire-8 questionnaire was completed by 110 patients (response rate, 50.2%). The median value of 28 indicates a high degree of patient satisfaction. An association between occurrence of major complications and patient satisfaction was seen. ConclusionsAesthetic surgery performed by plastic surgery residents under supervision by attending physicians is safe and provides for high levels of patient satisfaction postoperatively. Offering these services may be able to bridge the gap between providing high-quality aesthetic surgery training while yet recruiting an increasing number of patients who may appreciate the lower fees associated with these services.

miR-126 modulates angiogenic growth parameters of peripheral blood endothelial progenitor cells.

Abstract Vascularization plays an important role in tissue engineering applications. It is known that implantation of differentiated endothelial cells or endothelial progenitor cells (EPCs) from cord blood (cbEPCs) gives rise to the formation of a complex functional neovasculature, whereas EPCs isolated from peripheral blood (pbEPCs) have a limited capability to form blood vessels upon implantation. MicroRNA-126 (miR-126) has been shown to have pro-angiogenic effects in vivo. In this study, we investigated whether modulation of miR-126 expression in pbEPCs may alter their angiogenic properties. Gain of function and loss of function experiments revealed that miR-126 has anti-angiogenic effects in pbEPCs. Overexpression of miR-126 resulted in decreased proliferation, migration, invasion and tube formation, while inhibition of miR-126 induced the opposite effects. However, modulation of miR-126 expression did not influence apoptotic susceptibility of pbEPCs. This study provides evidence that inhibition of miR-126 improves angiogenesis-related growth parameters in pbEPCs and may represent a therapeutic option to ameliorate the angiogenic and vasculogenic properties of pbEPCs.

Treatment of male genital lymphedema: an integrated concept.

Genital lymphedema is a debilitating condition. Currently, therapeutic concepts include conservative and surgical measures of genital volume reduction, but no therapy standards exist. The aim of this study was to analyze our experience with the treatment of male genital lymphedema and to describe our integrated concept, which combines perioperative complex decongestive physiotherapy (CDP) with surgical reduction. We conducted a retrospective study of 51 male patients who were treated in our clinic between 1998 and 2013, acquiring data on etiology, performed therapy, and complication rates. Forty-three (84%) of our patients suffered from primary and eight (16%) from secondary lymphedema. Two patients (4%) exhibited isolated penile affection and 22 (43%) isolated scrotal affection, while the majority (27 or 53%) exhibited penoscrotal lymphedema. All patients underwent inpatient CDP preceding and following surgery. Surgical therapy involved debulking the scrotum in 25 patients (49%), isolated penile reduction in two (4%), and penoscrotal reduction in 24 (47%). Hydrocoelectomy was performed in 22 (43%) patients. Following resection of the diseased tissue, the defects were closed primarily using adjacent healthy skin. There was no need for local flaps or skin grafts. Three patients suffered from complications requiring revision surgery: hematoma in two patients (4%) and wound dehiscence in one (2%). We measured the change in quality of life that was achieved by these procedures using the Glasgow Benefit Inventory questionnaire. The patients reported improvement in general functioning and physical health, whereas the social support score was largely unaffected by the procedure. Our results show that genital lymphedema patients can be reliably treated by combining conservative and surgical therapy. Prior conservative reduction of the genitals facilitates surgery and allows consistent results at a low complication rate, whereas surgical reduction serves to enhance and stabilize the results of conservative treatment.

Shoulder silhouette and axilla reconstruction with free composite elbow tissue transfer following interscapulothoracic amputation.

Interscapulothoracic amputation (ISTA) encompasses the removal of the upper limb, scapula and clavicle. As the reconstructive and oncologic limb-saving techniques improved, the indications for this formidable procedure decreased. However, it is still the appropriate procedure in cases with extensive oncologic or traumatic involvement of the shoulder girdle. Following ISTA, the surgeon is not only faced with a large defect but also with severe functional and aesthetic impairments. A solution to these problems is the immediate reconstruction with a free composite transfer from the amputated extremity. We successfully treated three oncologic cases and one traumatic case using this technique. The recipient vessels included the subclavian artery and vein in three cases and the internal thoracic vessels in one case. After a mean follow-up time of 4.5 years, two of the three tumour patients were free of recurrent disease. In all cases stable wound closure was achieved. Three out of four patients would opt for surgery again, in spite of the high occurrence of complications. One patient died after a 14-year event-free postoperative course. We conclude that the defect following ISTA can be successfully covered using a free composite tissue transfer of the amputated disease-free elbow and forearm, while simultaneously reconstructing the shoulder silhouette and axilla. This procedure reduces functional and aesthetic impairments and improves the quality of life.

Soft Tissue Sarcomas of the Arm – Oncosurgical and Reconstructive Principles within a Multimodal, Interdisciplinary Setting

Soft tissue sarcomas of the upper extremity represent a severe threat for the patient and a difficult task for the treatment team. Due to the complex anatomy of the arm, most sarcomas involve valuable functional structures. Nonetheless, a large portion of the patients can be treated in a limb-sparing manner, and surgery is the mainstay of local tumor control. This review gives an overview of the disease entities and their epidemiology, on necessary patient work-up, staging, and imaging modalities, as well as the importance of interdisciplinary decision-making. The surgical therapies and principles of tumor excision are outlined, as well as reconstructive options. Furthermore, adjuvant treatments are discussed with a special focus on the various application techniques for radiation therapy. In spite of established treatment algorithms, each case is an individual challenge and individually tailored therapy is required. This aspect is illustrated by presenting three comprehensive cases demonstrating useful strategies. A summary of the relevant literature is given.

Transcriptomic Changes in Osteoblasts Following Endothelial Cell‐Cocultivation Suggest a Role of Extracellular Matrix in Cellular Interaction

Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1,004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT‐PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. J. Cell. Biochem. 117: 1869–1879, 2016.

Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments

Abstract Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3′-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3′-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications.

Calmodulin Regulated Spectrin Associated Protein 1 mRNA is Directly Regulated by miR-126 in Primary Human Osteoblasts: miR-126 REGULATES CAMSAP1 mRNA

Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR‐126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR‐126 gain‐of‐function and loss‐of‐function experiments in hOBs followed by microarray analysis in order to identify targets of miR‐126. The transcript cluster IDs were sieved by applying cut‐off criteria and by selecting transcripts which were upregulated following miR‐126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR‐126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR‐126. In this study, we show that miR‐126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756–1763, 2017.

Increased differentiation and production of extracellular matrix components of primary human osteoblasts after cocultivation with endothelial cells: A quantitative proteomics approach: SIMUNOVIC et al.

Coculturing of bone‐forming and blood vessel‐forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue‐engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin‐3, and cyclin‐dependent kinase 6. The findings were confirmed by enzyme‐linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real‐time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in‐depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.