Adiponectin and leptin up-regulate extracellular matrix production by dermal fibroblasts

Adipocytes were recently shown to secrete adipocytokines, such as adiponectin and leptin, which may have an endocrine role. Subcutaneous adipose tissue lies just beneath the dermis, and dermal condition is correlated with body mass index (BMI). However, it is not clear whether adipocytokines released by adipocytes in subcutaneous adipose tissue influence the adjacent dermis. We found that human dermal fibroblasts express genes encoding receptors for adiponectin and leptin, and that those cytokines both significantly increase production of hyaluronic acid (HA), a major extracellular matrix component (ECM) of dermis, by dermal fibroblasts. This effect is accompanied with up-regulation of HA synthase 2 gene expression. Moreover, adiponectin significantly increases production of collagen, the most abundant component of ECM in dermis, by dermal fibroblasts. These results suggest that subcutaneous adipocytes influence dermal condition by up-regulating collagen and HA production by dermal fibroblasts via secretion of adiponectin and leptin.     

Characteristic Expression of Extracellular Matrix in Subcutaneous Adipose Tissue Development and Adipogenesis; Comparison with Visceral Adipose Tissue

Adipose tissue is a connective tissue specified for energy metabolism and endocrines, but functional differences between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have not been fully elucidated. To reveal the physiological role of SAT, we characterized in vivo tissue development and in vitro adipocyte differentiation. In a DNA microarray analysis of SAT and VAT in Wistar rats, functional annotation clusters of extracellular matrix (ECM)-related genes were found in SAT, and major ECM molecules expressed in adipose tissues were profiled. In a histological analysis and quantitative expression analysis, ECM expression patterns could be classified into two types: (i) a histogenesis-correlated type such as type IV and XV collagen, and laminin subunits, (ii) a high-SAT expression type such as type I, III, and V collagen and minor characteristic collagens. Type (i) was related to basal membrane and up-regulated in differentiated 3T3-L1 cells and in histogenesis at depot-specific timings. In contrast, type (ii) was related to fibrous forming and highly expressed in 3T3-L1 preadipocytes. Exceptionally, fibronectin was abundant in developed adipose tissue, although it was highly expressed in 3T3-L1 preadipocytes. The present study showed that adipose tissues site-specifically regulate molecular type and timing of ECM expression, and suggests that these characteristic ECM molecules provide a critical microenvironment, which may affect bioactivity of adipocyte itself and interacts with other tissues. It must be important to consider the depot-specific property for the treatment of obesity-related disorders, dermal dysfunction and for the tissue regeneration.     

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

Biomimetic injectable HUVEC‐adipocytes/collagen/alginate microsphere co‐cultures for adipose tissue engineering

Engineering adipose tissue that has the ability to engraft and establish a vascular supply is a laudable goal that has broad clinical relevance, particularly for tissue reconstruction. In this article, we developed novel microtissues from surface‐coated adipocyte/collagen/alginate microspheres and human umbilical vein endothelial cells (HUVECs) co‐cultures that resembled the components and structure of natural adipose tissue. Firstly, collagen/alginate hydrogel microspheres embedded with viable adipocytes were obtained to mimic fat lobules. Secondly, collagen fibrils were allowed to self‐assemble on the surface of the microspheres to mimic collagen fibrils surrounding the fat lobules in the natural adipose tissue and facilitate HUVEC attachment and co‐cultures formation. Thirdly, the channels formed by the gap among the microspheres served as the room for in vitro prevascularization and in vivo blood vessel development. The endothelial cell layer outside the microspheres was a starting point of rapid vascular ingrowth. Adipose tissue formation was analyzed for 12 weeks at 4‐week intervals by subcutaneous injection into the head of node mice. The vasculature in the regenerated tissue showed functional anastomosis with host blood vessels. Long‐term stability of volume and weight of the injection was observed, indicating that the vasculature formed within the constructs benefited the formation, maturity, and maintenance of adipose tissue. This study provides a microsurgical method for adipose regeneration and construction of biomimetic model for drug screening studies. Biotechnol. Bioeng. 2013; 110: 1430–1443. © 2012 Wiley Periodicals, Inc.     

Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types

Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.     

Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

The non‐destructive and label‐free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non‐contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400–1800 cm^–1), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof‐of‐principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically‐degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non‐destructive and non‐contact tool to probe ECM state in situ. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

Adipose-derived stromal cells: Their identity and uses in clinical trials, an update

In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc. ). Apart from a phase Ⅲ trial on the treatment of fistula,most of these are in phaseⅠand use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use.     

Is there evidence for excessive free radical production in vivo during ultrasound-assisted liposuction?

The surgical technique of ultrasound-assisted liposuction has become a standard procedure for the treatment of lipodystrophy. However, little is known about the impact of this therapy on fatty tissue on the molecular level. There are concerns about possible adverse effects related to the high-intensity ultrasound energy, because in vitro studies have shown a substantial generation of free radicals. In this study, the authors investigated whether ultrasound waves can create an excessive free radical production in vivo by measuring lipid peroxidation products in the form of malondialdehyde equivalents. For this purpose, the thiobarbituric acid-reactive substances (TBARS) assay was chosen. In this test, malondialdehyde, a major product of lipid peroxidation, reacts with thiobarbituric acid to produce a pink adduct that can be measured spectrophotometrically. The authors determined oxidation products in 28 aspirates of 17 treated patients before ultrasound-assisted liposuction (0 minutes) to establish a baseline concentration and at 2, 5, and 10 minutes after the treatment was begun. Median malondialdehyde concentration of the control group (conventional liposuction, 0 minutes) was 3.40 nmol of malondialdehyde per gram of adipose tissue. Median concentrations after 2, 5, and 10 minutes of ultrasound-assisted liposuction were 7.45 (n = 28), 8.84 (n = 21), and 4.07 (n = 8) nmol malondialdehyde per gram adipose tissue, respectively. The differences were not statistically significant. The data suggest that there is no excessive formation of lipid oxidation products in response to free radicals. The antioxidative capacity of adipose tissue does not seem to be overwhelmed by the standard application regimen of ultrasound-assisted liposuction.     

Human adipose CD34+CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi‐potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co‐expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34^?CD90^? cells and was able to differentiate in vitro into adipocytes (PPARγ⁺ and adiponectin⁺) and endothelial cells (CD31⁺VEGF⁺Flk1⁺). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34⁺/CD90⁺ stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34⁺/CD90 ASCs are extremely useful for regenerative medicine. J. Cell. Biochem. 114: 1039–1049, 2013. © 2012 Wiley Periodicals, Inc.     

Collagen-based cell migration models in vitro and in vivo

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.     

A familiar stranger:CD34 expression and putative functions in SVF cells of adipose tissue

Human adipose tissue obtained by liposuction is easily accessible and an abundant potential source of autologous cells for regenerative medicine applications. After digestion of the tissue and removal of differentiated adipocytes, the so-called stromal vascular fraction (SVF) of adipose, a mix of various cell types, is obtained. SVF contains mesenchymal fibroblastic cells, able to adhere to culture plastic and to generate large colonies in vitro , that closely resemble bone marrow-derived colony forming units-fibroblastic, and whose expanded progeny, adipose mesenchymal stem/stromal cells (ASC), show strong similarities with bone marrow mesenchymal stem cells. The sialomucin CD34, which is well known as a hematopoietic stem cell marker, is also expressed by ASC in native adipose tissue but its expression is gradually lost upon standard ASC expansion in vitro . Surprisingly little is known about the functional role of CD34 in the biology and tissue forming capacity of SVF cells and ASC. The present editorial provides a short introduction to the CD34 family of sialomucins and reviews the data from the literature concerning ex- pression and function of these proteins in SVF cells and their in vitro expanded progeny.     

Acute inflammation plays a limited role in the regulation of adipose tissue COL1A1 protein abundance

Obesity is an inflammatory condition that is also associated with increased extracellular matrix (ECM) gene expression. However, a direct link between adipose tissue inflammation and ECM gene expression has not been established. Therefore, we determined the effect of chronic inflammation induced by obesity and acute inflammation by lipopolysaccharide (LPS) challenge on ECM genes including biglycan (BGN), collagen 1A1 (COL1A1) and COL6A1, major ECM genes in adipose tissue. Male C57BL/6J mice fed either a control diet (10% fat calories) or a high-fat diet (HFD) (60% fat calories) for 6 weeks were treated with LPS or saline 24 h before sacrifice. Expression of ECM genes in the epididymal (EWAT) and subcutaneous adipose tissue (SWAT) was determined by RT-PCR and protein abundance by Western blotting. Human SWAT from lean and obese subjects was also analyzed. Increased messenger RNA (mRNA) expression of ECM genes BGN and COL1A1 was observed in the mouse EWAT after HFD (P<.05). However, reduced amount of COL1A1 protein was observed in EWAT of mice on HFD and in SWAT from obese human subjects. Acute inflammation induced BGN mRNA in EWAT, enhanced the gene expression of matrix metalloproteases (MMPs) 3 and 9. Acute inflammation also resulted in higher MMP9 gelatinolytic activity; however, this showed no association with COL1A1 protein abundance. Higher MMP2 expression in mice on HFD suggests its involvement in the reduction of COL1A1 protein abundance with HFD. Elevated MMP9 gelatinolytic activity in SWAT from obese humans indicates a prominent role for MMP9 in SWAT COL1A1 protein turnover in humans.     

Opposite effect of corticosteroids and long-acting beta(2)-agonists on serum- and TGF-beta(1)-induced extracellular matrix deposition by primary human lung fibroblasts

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and major structural lung tissue changes including increased extracellular matrix (ECM) deposition. Inhaled corticosteroids and long-acting beta(2)-agonists (LABA) are the basic treatment for both diseases, but their effect on airway remodeling remains unclear. In this study, we investigated the effect of corticosteroids and LABA, alone or in combination, on total ECM and collagen deposition, gene expression, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels by primary human lung fibroblasts. In our model, fibroblasts in 0.3% albumin represented a non-inflammatory condition and stimulation with 5% FCS and/or TGF-beta(1) mimicked an inflammatory environment with activation of tissue repair. FCS (5%) increased total ECM, collagen deposition, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels. In 0.3% albumin, corticosteroids reduced total ECM and collagen deposition, involving the glucocorticoid receptor (GR) and downregulation of collagen, heat shock protein 47 (Hsp47), and Fli1 mRNA expression. In 5% FCS, corticosteroids increased ECM deposition, involving upregulation of COL4A1 and CTGF mRNA expression. LABA reduced total ECM and collagen deposition under all conditions partly via the beta(2)-adrenergic receptor. In combination, the drugs had an additive effect in the presence or absence of TGF-beta(1) further decreasing ECM deposition in 0.3% albumin whereas counteracting each other in 5% FCS. These data suggest that the effect of corticosteroids, but not of LABA, on ECM deposition by fibroblasts is altered by serum. These findings imply that as soon as airway inflammation is resolved, long-term treatment with combined drugs may beneficially reduce pathological tissue remodeling.     

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low‐dose ultraviolet light irradiation

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild‐type and GzmB‐knockout mice were repeatedly exposed to minimal erythemal doses of solar‐simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild‐type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB‐mediated fibronectin fragments increased the expression of collagen‐degrading matrix metalloproteinase‐1 (MMP‐1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age‐related chronic inflammatory diseases.     

2,2′,4,4′,5‐Pentabromodiphenyl ether induces lipid accumulation throughout differentiation in 3T3‐L1 and human preadipocytes in vitro

Flame retardants, specifically polybrominated diphenyl ethers (PBDEs), are chemical compounds widely used for industrial purposes and household materials. NHANES data indicate that nearly all Americans have trace amounts of PBDEs in serum, with even higher levels associated with occupational exposure. PBDEs are known to bioaccumulate in the environment due to their lipophilicity and stability, and more importantly, they have been detected in human adipose tissue. The present study examined whether the PBDE congener, BDE‐99 (2,2′,4,4′,5‐pentabromodiphenyl ether; 0.2‐20 μM), enhances the adipogenesis of mouse and human preadipocyte cell models in vitro via induced lipid accumulation. 3T3‐L1 mouse preadipocytes and human visceral preadipocytes demonstrated enhanced hormone‐induced lipid accumulation upon BDE‐99 treatment. In addition, BDE‐99 (20 μM) induced preadipocyte differentiation and lipid development in nondifferentiated human preadipocytes. BDE‐99, the second most abundant congener in human adipose tissue, increased total lipids in differentiating adipocytes and therefore showed a potential role in the regulation of adipogenesis. This warrants more research to further understand the impact of lipophilic persistent pollutants on adipose tissue homeostasis.     

Adipogenic differentiation of adipose tissue derived adult stem cells in nude mouse

We evaluated the use of a combination of adipose tissue derived adult stem cells (ADSCs) obtained from liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for adipose tissue engineering. Adipogenesis was examined in nude mice injected subcutaneously with ADSCs (group I), PLGA spheres (group II), or ADSCs attached PLGA spheres (group III) cultured in adipogenic medium for 7 days. After 4 and 8 weeks, newly formed adipose tissue was observed in groups II and III but not in group I. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration and adipogenic differentiation in group III than in group II. RT-PCR confirmed that, after 8 weeks, the PLGA-attached ADSCs had fully differentiated into adipocytes. This study provides significant evidence that ADSCs and PLGA spheres can be used in a clinical setting to generate adipose tissue as a noninvasive soft tissue filler.