Integrin alpha V and alpha 5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

Die Physiologie von humanen adipozytären Vorläuferzellen (ASC) wird größtenteils durch biochemische und mechanische Einflüsse aus der extrazellulären Matrix (EZM) gesteuert, welche von Integrinen aufgenommen und übermittelt werden. Man weiß, dass spezifische EZM Bestandteile die Proliferation und Differenzierung von ASC beeinflussen. Allerdings ist das Wissen um die genauen Mechanismen hier noch stark beschränkt. In dieser Arbeit wurde eine Genexpressionsanalyse von 18 Integrin alpha-Subtypen in gesorteten ASC und Adipozyten durchgeführt. Es konnten damit die Repression der RDG-Motiv spezifischen Integrine Integrin-alpha-V (ITGAV) und Integrin-alpha-5 sowie eine erhöhte Expression von Laminin bindenden Integrinen und Leukozyten-spezifischen Integrinen sowie individuelle Regulationsmuster für Kollagen- und LDV-Motiv bindende Integrine in der Adipogenese gezeigt werden. Ein Knock-down von ITGAV reduzierte -im Gegensatz zum Knock-down von ITGA5- die Proliferation, induzierte p21 (Cip1), reprimierte Survivin und zeigte darüberhinaus eine spezifische Regulation des Mediators TAZ im Hippo-Signalweg. Der Knock-down von beiden Integrinen führte zu verstärkter Adipogenese während eine Überexpression diese unterdrückte. Die Inhibition von ITGA mit dem pharmakologischen Wirkstoff Cilengitide zeigte einen vergleichbaren Phänotyp. Zusammenfassen lässt sich sagen, dass ASC spezifische Expressionsmuster für Integrine aufweisen. Wir konnten sowohl für ITGAV als auch ITGA5 eine spezifische Rolle in der ASC Physiologie definieren, insbesondere einen negativen Einfluss der RGD-Motiv mediierten Integrin-Signaltransduktion auf die Adipogenese.The fate of human adipose tissue stem cells (ASC) is largely determined by biochemical and mechanical cues from the extracellular matrix (ECM), which are sensed and transmitted by integrins. It is well known that specific ECM constituents influence ASC proliferation and differentiation. Nevertheless, knowledge on how individual integrins regulate distinct processes is still limited. We performed gene profiling of 18 alpha integrins in sorted ASC and adipocytes, identifying downregulations of RGD-motif binding integrins integrin-alpha-V (ITGAV) and integrin-alpha-5 (ITGA5), upregulation of laminin binding and leukocyte-specific integrins and individual regulations of collagen and LDV-receptors in differentiated adipocytes in-vivo. Gene function analyses in in-vitro cultured ASC unraveled differential functions of ITGA5 and ITGAV. Knockdown of ITGAV, but not ITGA5 reduced proliferation, caused p21(Cip1) induction, repression of survivin and specific regulation of Hippo pathway mediator TAZ. Gene knockdown of both integrins promoted adipogenic differentiation, while transgenic expression impaired adipogenesis. Inhibition of ITGAV using cilengitide resulted in a similar phenotype, mimicking loss of pan-ITGAV expression using RNAi. Herein we show ASC specific integrin expression patterns and demonstrate distinct regulating roles of both integrins in human ASC and adipocyte physiology suggesting a negative impact of RDG-motif signaling on adipogenic differentiation of ASC via ITGA5 and ITGAV.submitted by: Dr. med. univ. Evi M. MorandiMedizinische Universität Innsbruck, Dissertation, 2017OeBB(VLID)172468

Resection of Skin Cancer Resulting in Free Vascularized Tissue Reconstruction: Always a Therapeutic Failure?

The globally increasing incidence of cutaneous malignancies leads, in parallel, to increasing numbers of locally advanced skin cancer resulting in reconstructive surgery. Reasons for locally advanced skin cancer may be a patient’s neglect or aggressive tumor growth, such as desmoplastic growth or perineural invasion. This study investigates characteristics of cutaneous malignancies requiring microsurgical reconstruction with the aim of identifying possible pitfalls and improving diagnostic and therapeutic processes. A retrospective data analysis from 2015 to 2020 was conducted. Seventeen patients (n = 17) were included. The mean age at reconstructive surgery was 68.5 (±13) years. The majority of patients (14/17, 82%) presented with recurrent skin cancer. The most common histological entity was squamous cell carcinoma (10/17, 59%). All neoplasms showed at least one of the following histopathological characteristics: desmoplastic growth (12/17, 71%), perineural invasion (6/17, 35%), or tumor thickness of at least 6 mm (9/17, 53%). The mean number of surgical resections until cancer-free resection margins (R0) were achieved was 2.4 (±0.7). The local recurrence rate and the rate of distant metastases were 36%. Identified high-risk neoplastic characteristics, such as desmoplastic growth, perineural invasion, and a tumor depth of at least 6 mm, require a more extensive surgical treatment without concerns about defect size

Long-Term Results after Autologous Breast Reconstruction with DIEP versus PAP Flaps Based on Quality of Life and Aesthetic Outcome Analysis

(1) Background: This work aimed to conduct a comparative study, providing long-term data about patient-reported outcome measures and donor site scar assessments, as well as an aesthetic evaluation of the reconstructed breasts in patients with DIEP versus PAP flap breast reconstruction. (2) Methods: This prospective, single-center, matched cohort study included a total of 36 patients after DIEP and PAP flap breast reconstruction. The evaluation was carried out using the Breast-Q and POSAS questionnaire, as well as the Breast Aesthetic Scale for cosmetic analysis, by four plastic surgeons. (3) Results: The postoperative Breast-Q evaluation revealed no significant differences between both patient groups for the categories of the physical well-being of the donor site, the physical well-being of the breast, and satisfaction with the breast. A scar evaluation of the donor site region showed equivalent results for the thigh and abdomen regions, concerning the overall opinion of the patients and the observers. There was no significant difference between both methods of reconstruction for all aspects of breast aesthetics. (4) Conclusions: Similar results for donor site morbidity, scar quality, and the aesthetic outcome of the breasts in both the DIEP and PAP patient groups have been demonstrated. Hence, in those cases suitable for both types of reconstruction, the decision can be based on factors such as patients’ lifestyles, leisure activities, and preferences

Donor-Site Morbidity and Quality of Life after Autologous Breast Reconstruction with PAP versus TMG Flap

The transverse myocutaneous gracilis (TMG) and the profunda artery perforator (PAP) flap are both safe choices for autologous breast reconstruction originating from the same donor region in the upper thigh. We aimed to compare the post-operative outcome regarding donor-site morbidity and quality of life. We included 18 patients who had undergone autologous breast reconstruction with a PAP flap (n = 27 flaps). Prospective evaluation of donor-site morbidity was performed by applying the same questionnaire that had already been established in a previous study evaluating TMG flap (n = 25 flaps) outcome, and results were compared. Comparison of the two patient groups showed equivalent results concerning patient-reported visibility of the donor-site scar and thigh symmetry. Still, the TMG group was significantly more satisfied with the scar (p = 0.015) and its position (p = 0.001). No difference was found regarding the ability to sit for prolonged periods. Donor-site wound complications were seen more frequently in the PAP group (29.6%) than in the TMG group (4.0%). Both groups expressed rather high satisfaction with their quality of life. Both flaps show minimal functional donor-site morbidity and high patient satisfaction. To minimize wound healing problems in PAP patients, thorough planning of the skin paddle is necessary

Differentiation between Acute Skin Rejection in Allotransplantation and T-Cell Mediated Skin Inflammation Based on Gene Expression Analysis

Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction. However, skin rejection and side effects of long-term immunosuppression still remain a major hurdle for wide adoption of this excellent reconstructive technique. Histopathologic changes during acute skin rejection in vascular composite allotransplantation often mimic inflammatory skin disorders and are hard to distinguish. Hence, the identification of diagnostic and therapeutic markers specific for skin rejection is of particular clinical need. Here we present novel markers allowing for early differentiation between rejection in hind limb allotransplantation and contact hypersensitivity. Assessment of Ccl7, Il18, and Il1b expression is most indicative of distinguishing skin rejection from skin inflammatory disorders. Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration. Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type. In synopsis of the RNA expression profile and previously assessed protein expression, the Il1 family appears as a promising option for accurate skin rejection diagnosis and, as a following step, for development of novel rejection treatments

Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing

Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization

Abstract Background Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. Methods We examined CD45−/CD31−/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. Results CD45−/CD31−/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. Conclusion Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing