MicroRNA-145 Regulates the Differentiation of Adipose Stem Cells Toward Microvascular Endothelial Cells and Promotes Angiogenesis.

RATIONALE: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in ischemic tissues. However, the mechanism that promotes ASCs differentiation toward endothelial cells (ECs) is not known. OBJECTIVE: To investigate the mechanisms of ASCs differentiation into ECs. METHODS AND RESULTS: ASCs were isolated from clinical lipoaspirates and cultured with DMEM or endothelial cell-conditioned medium. Endothelial cell-conditioned medium induced downregulation of miR-145 in ASCs and promoted endothelial differentiation. We identified bFGF (basic fibroblast growth factor) released by ECs as inducer of ASCs differentiation through receptor-induced AKT (protein kinase B) signaling and phosphorylation of FOXO1 (forkhead box protein O1) suppressing its transcriptional activity and decreasing miR-145 expression. Blocking bFGF-receptor or PI3K/AKT signaling in ASCs increased miR-145 levels. Modulation of miR-145 in ASCs, using a miR-145 inhibitor, regulated their differentiation into ECs: increasing proliferation, migration, inducing expression of EC markers (VE-cadherin, VEGFR2 [vascular endothelial growth factor receptor 2], or VWF [von Willebrand Factor]), and tube-like formation. Furthermore, in vivo, downregulation of miR-145 in ASCs enhanced angiogenesis in subcutaneously implanted plugs in mice. In a murine hindlimb ischemia model injection of ASCs with downregulated miR-145 induced collateral flow and capillary formation evidenced by magnetic resonance angiography. Next, we identified ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) as the target of miR-145. Upregulation of miR-145 in ASCs, by mimic miR-145, suppressed ETS1 expression and consequently abolished EC differentiation and the angiogenic properties of endothelial cell-conditioned medium-preconditioned ASCs; whereas, overexpression of ETS1 reversed the abrogated antiangiogenic capacity of miR-145. ETS1 overexpression induced similar results to those obtained with miR-145 knockdown. CONCLUSIONS: bFGF released by ECs induces ASCs differentiation toward ECs through miR-145-regulated expression of ETS1. Downregulation of miR-145 in ASCs induce vascular network formation in ischemic muscle.

Differentiation of Adipose Tissue Mesenchymal Stem Cells into Endothelial Cells Depends on Fat Depot Conditions: Regulation by miRNA.

The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or reduce in size throughout the adult lifespan, which is linked to the development of its vasculature. This increase in AT vasculature could be mediated by the differentiation of adipose tissue-derived stem cells (ASCs) into endothelial cells (ECs) and form new microvasculature. We have already shown that microRNA (miRNA)-145 regulates the differentiation of ASCs into EC-like (ECL) cells. Here, we investigated whether ASCs-differentiation into ECs is governed by a miRNAs signature that depends on fat depot location and /or the metabolic condition produced by obesity. Human ASCs, which were obtained from white AT by surgical procedures from lean and obese patients, were induced to differentiate into ECL cells. We have identified that miRNA-29b-3p in both subcutaneous (s)ASCs and visceral ASCs and miRNA-424-5p and miRNA-378a-3p in subcutaneous (s)ASCs are involved in differentiation into EC-like cells. These miRNAs modulate their pro-angiogenic effects on ASCs by targeting FGFR1, NRP2, MAPK1, and TGF-ß2, and the MAPK signaling pathway. We show for the first time that miRNA-29b-3p upregulation contributes to ASCs' differentiation into ECL cells by directly targeting TGFB2 in both sASCs and visceral ASCs. Moreover, our results reveal that, independent of sASCs' origin (obese/lean), the upregulation of miRNA-378a-3p and the downregulation of miRNA-424-5p inhibit MAPK1 and overexpress FGFR1 and NRP2, respectively. In summary, both the adipose depot location and obesity affect the differentiation of resident ASCs through the expression of specific miRNAs.

Stem cells isolated from adipose tissue of obese patients show changes in their transcriptomic profile that indicate loss in stemcellness and increased commitment to an adipocyte-like phenotype.

BACKGROUND: The adipose tissue is an endocrine regulator and a risk factor for atherosclerosis and cardiovascular disease when by excessive accumulation induces obesity. Although the adipose tissue is also a reservoir for stem cells (ASC) their function and "stemcellness" has been questioned. Our aim was to investigate the mechanisms by which obesity affects subcutaneous white adipose tissue (WAT) stem cells. RESULTS: Transcriptomics, in silico analysis, real-time polymerase chain reaction (PCR) and western blots were performed on isolated stem cells from subcutaneous abdominal WAT of morbidly obese patients (ASCmo) and of non-obese individuals (ASCn). ASCmo and ASCn gene expression clustered separately from each other. ASCmo showed downregulation of "stemness" genes and upregulation of adipogenic and inflammatory genes with respect to ASCn. Moreover, the application of bioinformatics and Ingenuity Pathway Analysis (IPA) showed that the transcription factor Smad3 was tentatively affected in obese ASCmo. Validation of this target confirmed a significantly reduced Smad3 nuclear translocation in the isolated ASCmo. CONCLUSIONS: The transcriptomic profile of the stem cells reservoir in obese subcutaneous WAT is highly modified with significant changes in genes regulating stemcellness, lineage commitment and inflammation. In addition to body mass index, cardiovascular risk factor clustering further affect the ASC transcriptomic profile inducing loss of multipotency and, hence, capacity for tissue repair. In summary, the stem cells in the subcutaneous WAT niche of obese patients are already committed to adipocyte differentiation and show an upregulated inflammatory gene expression associated to their loss of stemcellness.

The subcutaneous adipose tissue reservoir of functionally active stem cells is reduced in obese patients.

It has been demonstrated that the adipose tissue, a highly functional metabolic tissue, is a reservoir of mesenchymal stem cells. The potential use of adipose-derived stem cells (ADSCs) from white adipose tissue (WAT) for organ repair and regeneration has been considered because of their obvious benefits in terms of accessibility and quantity of available sample. However, the functional capability of ADSCs from subjects with different adiposity has not been investigated. It has been our hypothesis that ADSCs from adipose tissue of patients with metabolic syndrome and high adiposity may be functionally impaired. We report that subcutaneous WAT stromal vascular fraction (SVF) from nonobese individuals had a significantly higher number of CD90+ cells than SVF from obese patients. The isolated ADSCs from WAT of obese patients had reduced differentiation potential and were less proangiogenic. Therefore, ADSCs in adipose tissue of obese patients have lower capacity for spontaneous or therapeutic repair than ADSCs from nonobese metabolically normal individuals.

Distally based dorsal nasal flap in nasal ala reconstruction: anatomic study and clinical experience.

BACKGROUND: The nasal dorsum is a good skin flap donor site for alar reconstructions because of its qualities: appropriate color, texture, and thickness. OBJECTIVE: An anatomic vascular study on cadaver and the clinical use of the dorsal nasal skin flap, inferiorly based on the nasal septal branches, is reported. MATERIALS AND METHODS: Vascular anatomy of the nasal dorsum was demonstrated in five fresh-frozen latex-injected heads. Fourteen patients were operated of reconstruction of the nasal ala using an inferiorly based dorsal nasal flap. RESULTS: Nasal septal branches, from the superior labial arteries, give vascular supply to the nasal tip. Connections of these arteries with lateral nasal branches (facial system) and dorsal nasal arteries (ophthalmic system) form a consistent vascular network in the dorsal nasal superficial muscular aponeurotic system and allow to safely raise cutaneous flaps distally based. No total or partial loss of the flaps was observed in clinical use. The donor site was sutured directly in 13 patients and with a skin graft in one. CONCLUSION: The inferiorly based dorsal nasal flap provides very good cosmetic and functional results and could be considered an additional adequate surgical option for nasal ala reconstruction, especially when skin from the nasolabial fold, upper lip, and cheek is not available. The authors have indicated no significant interest with commercial supporters.

Preoperative digital three-dimensional planning for rhinoplasty.

BACKGROUND: This report describes preoperative digital planning for rhinoplasty using a new three-dimensional (3D) radiologic viewer that allows both patients and surgeons to visualize on a common monitor the 3D real aspect of the nose in its inner and outer sides. METHODS: In the period 2002 to 2008, 210 patients underwent rhinoplasty procedures in the authors' clinic. The patients were randomly divided into three groups according to the type of preoperative planning used: photos only, a simulated result by Adobe Photoshop, or the 3D radiologic viewer. The parameters evaluated included the number of patients that underwent surgery after the first consultation, the number of patients who asked for a reintervention, patient satisfaction (according to a test given to the patients 12 months postoperatively), the surgical time required for a functional intervention, and the improvement in nasal function by postoperative rhinomanometry and subjective evaluation. RESULTS: Computer-aided technologies led to a higher number of patients deciding to undergo a rhinoplasty. Simulation of the postoperative results was not as useful in the postoperative period due to the higher number of reintervention requests. CONCLUSION: The patients undergoing rhinoplasties preferred new technologies in the preoperative period. The advantages of using the 3D radiologic viewer included improved preoperative planning, reduction in intraoperative stress, a higher number of patients undergoing surgery, reduction in postoperative surgical corrections, reduction in surgical time for the functional intervention, a higher rate of improvement in nasal function, a higher percentage of postoperative satisfaction, and reduced costs.

Gluteal augmentation with cryopreserved fat.

Gluteal augmentation with autologous fat is becoming a standard ancillary procedure for sculpting the buttock area. The high rate of resorption due to aggressive harvesting techniques or inadequate injection procedures often leads to repeated treatments. Currently, several techniques for storing fat by controlled freezing and thawing procedures can guarantee a high rate of cell viability, similar to that obtained with fresh tissue. This allows surgeons to compile fat tissue available for future repeat injections, decreasing additional costs and morbidity for patients. The authors describe a case of gluteal augmentation with cryopreserved fat in a 42-year-old man.

Cardiovascular Risk Factors and Differential Transcriptomic Profile of the Subcutaneous and Visceral Adipose Tissue and Their Resident Stem Cells.

BACKGROUND: The increase in the incidence of obesity and obesity-related cardiovascular risk factors (CVRFs) over the last decades has brought attention on adipose tissue (AT) pathobiology. The expansion of AT is associated with the development of new vasculature needed to perfuse the tissue; however, not all fat depots have the same ability to induce angiogenesis that requires recruitment of their own endothelial cells. In this study we have investigated the effect of different CVRFs, on the angiogenic capacity of the subcutaneous (SAT) and visceral (VAT) adipose tissue and on the function of their mesenchymal cell reservoir. METHODS: A transcriptomic approach was used to compare the different angiogenic and inflammatory profiles of the subcutaneous and visceral fat depots from individuals with obesity, as well as their resident stem cells (ASCs). Influence of other risk factors on fat composition was also measured. Finally, the microvesicles (MVs) released by ASCs were isolated and their regenerative potential analyzed by molecular and cellular methodologies. RESULTS: Obesity decreases the angiogenic capacity of AT. There are differences between SAT and VAT; from the 21 angiogenic-related genes analyzed, only three were decreased in SAT compared with those decreased in VAT. ASCs isolated from both fat depots showed significant differences; there was a significant up-regulation of the VEGF-pathway on visceral derived ASCs. ASCs release MVs that stimulate endothelial cell migration and angiogenic capacity. CONCLUSIONS: In patients with obesity, SAT expresses a greater number of angiogenic molecules than VAT, independent of the presence of other CVRFs.

The 'propeller' distal anteromedial thigh perforator flap. Anatomic study and clinical applications.

BACKGROUND: The leg and peripatellar region have always been known as a poor source of available flaps. One flap donor site that has proven to be adequate is the distal anteromedial half of the thigh. Due to the potential and plentiful vascular sources of this anatomic region we decided to study the distal anteromedial thigh and its clinical applications. ANATOMIC STUDY: Sixteen cryopreserved inferior limbs were latex-injected in the femoral artery and the skin perforators of the distal anteromedial thigh and their source vessels were studied. CLINICAL STUDY: In a period between December 2000 and June 2005, skin islands from the distal anteromedial aspect of the thigh of six patients were transferred, as local perforator flaps, to reconstruct the peripatellar region and upper leg soft tissue defects. Every flap was based on a single adequate perforator vessel. The tissue was rotated, as a 'propeller', through 180 degrees and the flap was named 'the propeller distal anteromedial thigh perforator flap'. RESULTS: In the distal anteromedial thigh the anatomic variability includes not only perforator vessels but also their source vessels. Skin perforators can come from each of the deep vessels. Our clinical results, with a follow up of 1-4 years, show no total flap losses. Partial necrosis > 20% happened in one diabetic patient. CONCLUSION: The propeller distal anteromedial thigh perforator flap can be reliably transferred based on only one adequate perforator vessel. It reduces the morbidity and improves the availability of the distal anteromedial thigh as a flap donor site and represents an additional reconstructive option for knee and upper leg defects.

Our experience in lower limb reconstruction with perforator flaps.

The application of Taylor's concept about body angiosomes, referred to tissue transfers, has meant that the development of the perforator flaps and muscles is no longer needed as a carrier of skin flap vascularity. In this paper, we revise 59 lower limb reconstructions with local and free perforator flaps performed in the last 5 years, and a basic reconstructive algorithm is also suggested to help with the management of the lower limb soft tissue reconstruction with perforator flaps. The advantages of the perforator flaps are (1) muscles and their function are preserved; (2) the main vascular trunks are spared; (3) it is possible to make a more specific reconstruction, replacing "like with like" (even performing compound or chimeric flaps); (4) the donor site can often be closed primarily; (5) the general morbidity is reduced; (6) a better cosmetic result can be achieved.