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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 7 2609-2614
Copyright © 2000 by The Endocrine Society

Original Studies

Autocrine Regulation of Human Preadipocyte Migration by Plasminogen Activator Inhibitor-1

David L. Crandall, Dennis E. Busler, Barbara McHendry-Rinde, Thomas M. Groeling and John G. Kral

Wyeth Research, Radnor, Pennsylvania 19087; and Department of Surgery, Downstate Medical Center (J.G.K.), Brooklyn, New York 11203

Address all correspondence and requests for reprints to: Dr. David L. Crandall, Wyeth Research, P.O. Box 42528, Philadelphia, Pennsylvania 19101. E-mail: crandad{at}war.wyeth.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the initial stages of adipogenesis is migration of preadipocytes of mesenchymal origin into cell clusters to form primitive fat organs. The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is synthesized and released from human adipose tissue ex vivo and regulates smooth muscle and endothelial cell migration in vitro, but its role in adipose tissue is not known. We investigated the role of PAI-1 in cultures of human preadipocytes from men and women of various ages and body mass indexes. Human preadipocytes expressed the messenger ribonucleic acid for PAI-1 and released significant quantities of PAI-1 protein into the medium. As PAI-1 regulates motility through the interaction of vitronectin with its receptor, the integrin {alpha}Vß3, we identified this receptor in human preadipocytes. Flow cytometric analysis indicated that human preadipocytes express the vitronectin receptor {alpha}Vß3 in a similar pattern as human umbilical vein endothelial cells. Functional studies indicated that active, but not latent, PAI-1 inhibited preadipocyte attachment to vitronectin with an IC50 of 13.3 nmol/L, and preincubation of vitronectin-coated Transwells with active PAI-1 prevented preadipocyte migration. Vitronectin was identified in homogenates of the stromal-vascular fraction of human adipose tissue, but was absent from human adipocytes and cultured preadipocytes. These data indicate that human preadipocyte migration is regulated through the endogenous expression of PAI-1 and {alpha}Vß3 integrin, a novel autocrine mechanism for potentially regulating cell cluster formation in adipogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PLASMINOGEN activator inhibitor (PAI-1), a member of the serine protease inhibitor family, inhibits both tissue-type plasminogen and urokinase type plasminogen activators. PAI-1 inactivation of the fibrinolytic system prevents fibrin degradation, and elevated circulating PAI-1 increases the risk for thrombosis (1, 2). Plasma PAI-1 is increased with obesity (3) and correlates with the amount of visceral adipose tissue (4). PAI-1 is synthesized by and released from cultured murine adipocytes (5), and human adipose tissue explants maintained under specific, short term culture conditions express PAI-1 messenger ribonucleic acid (mRNA) (6).

More recent investigations have identified another role for PAI-1 in cell migration and angiogenesis. PAI-1 has been shown to affect cell migration by competing for a specific integrin-binding site on vitronectin (7), and PAI-1 knockout mice are unable to vascularize tumors (8). The integrin for which PAI-1 competes, {alpha}Vß3, has been shown to be critical to developmental angiogenesis (9), and PAI-1 binding to the extracellular matrix may function to regulate local pericellular proteolysis central to tissue remodeling. Additionally, PAI-1 knockout mice crossed with genetically obese mice weigh significantly less than wild-type obese mice (10), although the mechanism of action of this effect is unknown.

Obesity is a disease characterized by an increased fat mass, but because of the belief that the number of fat cells is fixed in adulthood (11), studies of adipose tissue growth have focussed almost exclusively on the enlargement of the adipocyte once the tissue matrix is established. Importantly, however, studies of the relationship between fat mass and adipocyte number in humans clearly indicate the potential for adipocyte hyperplasia in adulthood in the range of billions of fat cells (12, 13). Similar observations of adipose tissue growth through adipocyte hyperplasia are documented for animal models of obesity (14).

Investigations into the early stages of human adipose tissue development are primarily descriptive anatomical studies in human fetuses. The process of adipose tissue organogenesis is characterized by the formation of a vascularized primitive fat organ, followed by migration of preadipocytes into defined fat cell clusters (15), but the molecules regulating this process are unknown. The current study investigates novel aspects of cell physiology in human adipose tissue primary cultures, focussing specifically on identifying the various components required to establish PAI-1 as a regulatory protein in human preadipocyte migration, potentially contributing to adipose tissue growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of human preadipocytes

Human preadipocytes were cultured from sc biopsies as previously described in detail (16). Cultures were maintained in a growth medium consisting of medium 199, 10% heat-inactivated FCS, and 1% antibiotic-antimycotic (Life Technologies, Inc., Grand Island, NY) at 37 C in 5%CO2. The human preadipocytes stain negative for factor VIII, and when stimulated to differentiate into the adipocyte phenotype, they express leptin mRNA and increased staining for triglyceride (16).

Human adipose tissue homogenates

Subcutaneous or mesenteric (visceral) adipose tissue was obtained by knife biopsy or suction-assisted sc lipectomy from men and women varying in age between 22–75 yr, with body mass indexes (BMIs) of 19–42 kg/m2. None of the individuals was diabetic or had undergone significant weight loss preoperatively. Samples were quickly frozen and maintained at -80 C until analysis. Both types of samples were thawed the day of the experiment and subjected to collagenase digestion and selective filtration (16). For liposuction samples, this procedure resulted predominantly in an adipocyte fraction only, whereas for knife biopsies, a significant stromal-vascular fraction was obtained as well. After washing with sterile phosphate-buffered saline (PBS), tissue fractions were homogenized briefly in a lysis buffer consisting of 1 x PBS, 1% Triton, and 1% Nonidet P-40 (Sigma, St. Louis, MO) and spun at 10,000 x g for 15 min, and the supernatant was used for determination of vitronectin content.

Identification of components of the PAI-1 migration regulatory pathway

Primary cultures of human preadipocytes were transferred to 24-well plates in passages 2–4, and after attachment, fresh medium containing 10% serum was added, with PAI-1 antigen measured in the conditioned medium 24 h later by enzyme-linked immunosorbent assay (American Diagnostica, Greenwich, CT).

Total RNA was prepared from cultured human preadipocytes using an adaptation of the guanidinium thiocyanate extraction method (RNeasy, QIAGEN, Chatsworth, CA). RT-PCR was performed for identification of PAI-1 mRNA (17), with RT-PCR of mRNA for human ß2-microglobulin (CLONTECH Laboratories, Inc., Palo Alto, CA) determined from the same complementary DNA. In addition to cultured preadipocytes, adipocyte RNA was obtained from the adipocyte fraction of human adipose tissue after collagenase digestion, and RT-PCR was employed for the identification of vitronectin mRNA using the procedure described by Seiffert et al. (18). Human liver RNA was obtained from CLONTECH Laboratories, Inc. and served as the standard for vitronectin mRNA identification.

Identification of the {alpha}Vß3 integrin on human preadipocytes used flow cytometric analysis. For flow cytometry, preadipocytes were detached using cell dissociation buffer (Life Technologies, Inc., Gaithersburg, MD). Cell suspensions were pelleted, washed with 10 mL HBSS, and resuspended in PBS containing 2% heat-inactivated FBS. Cell suspensions (50 µL; 5 x 105 cells) were stained using 50 µL anti-{alpha}Vß3 monoclonal antibody (CD51/61, PharMingen, San Diego, CA) diluted to 10 µg/mL, followed by staining with a 1:100 dilution of goat antimouse secondary antibody directly conjugated to phycoerythrin (Immunotech, Westbrook, ME). Cells were resuspended in 2 µg/mL propidium iodide/PBS immediately before flow cytometric analysis. Collection criteria based on propidium iodide exclusion were used to acquire data from 5000 vital cells/sample (FACSCalibur flow cytometer, Becton Dickinson and Co., San Jose, CA). Human preadipocytes stained with the isotypic (IgG1{kappa}) antibody (Sigma) served as controls.

Identification and quantification of vitronectin

For identification of vitronectin in cultured cells and in adipose tissue biopsies we employed a capture antibody assay for native vitronectin. A 96-well plate was coated overnight at 4 C with 100 µL of a 10 µg/mL solution of vitronectin capture antibody MA-16A7 (Molecular Innovations, Inc., Royal Oak, MI). The plate was washed 3 times with Tris-buffered saline Triton (0.05%) (TBST) and blocked for 1 h with 300 µL blocking buffer (3% albumin/TBST). The blocking buffer was aspirated, and 25 µL sample (cultured preadipocyte or adipose tissue fraction homogenate) or standard were added to the plate (standards of human native vitronectin from 0.5–200 ng/mL). The plates were incubated for 30 min at 25 C with gentle shaking, followed by washing 3 times in TBST. A 100-µL solution of rabbit antihuman vitronectin polyclonal antibody diluted 1:1,000 in blocking buffer was added, followed by incubation for 30 min with gentle shaking. After washing 3 times with TBST, 100 µL biotinylated goat anti-rabbit IgG (1:50,000 in Tris-buffered saline) was added to each well, then incubated for 30 min while shaking. After aspiration, 100 µL of a solution of avidin alkaline phosphatase (1:10,000 dilution in blocking buffer) were added, followed by a 30-min incubation. All wells were aspirated and washed 3 times with TBST, and 150 µL of a 0.1% PNPP solution were added. After incubation for 1 h, absorbance was measured at 405 nm. Fresh human citrated plasma was used to compare the vitronectin values obtained for adipose tissue to those in the circulation. In addition, human artery and vein samples were homogenized and used for determination of vitronectin in the vasculature.

Attachment and migration of human preadipocytes

Preadipocytes were trypsinized, washed in PBS, and resuspended in phenol red-free medium containing 0.5% heat-inactivated FCS. Preadipocytes (1 x 105/mL) were preincubated at room temperature for 15 min with varying concentrations (5–100 nmol/L) of active human PAI-1 (Molecular Innovations) solubilized in 50 mmol/L phosphate buffer (pH 6.6). Cells were subsequently incubated for 1 h at 37 C on a vitronectin (Molecular Innovations)-coated (1 µg/mL) 96-well plate (7). At the end of the incubation period, the plate was rinsed with PBS, followed by assessment of cell attachment using a chromogenic reporter (CellTiter, Promega Corp., Madison, WI). The percentage of attached cells was determined, as was the concentration of PAI-1 required for 50% inhibition of attachment. In a separate set of experiments, preadipocyte migration assays were performed using a Transwell system with a 3-µm pore size in a 6.5-mm insert (Costar, Cambridge, MA). The insert was coated with human vitronectin as previously described by Stefansson and Lawrence (7). For determination of the effect of PAI-1 on cell attachment, either 1 µmol/L active or latent PAI-1 (pH 6.6) was added to the vitronectin-coated insert for 15 min at 37 C, followed by the addition of 50,000 human preadipocytes suspended in medium 199/0.5% FCS. The insert was placed in a tissue culture well containing medium 199/10% FCS, creating a chemotactic serum gradient compared to the insert. The culture was incubated at 37 C for 1 h, the insert was removed, and cells were fixed and stained with Hema 3 stain (Fisher Scientific, Pittsburgh, PA). After microscopic imaging, the number of cells that had migrated through the Transwell were quantitated using imaging software (Noesis Vision, Inc., Montréal, Canada).

Statistical analysis

Statistical analysis was performed using the Kruskal-Wallis analysis for multiple groups. Group means ± SEM were considered significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial experiments were performed to determine the expression of PAI-1 mRNA and protein by cultured human preadipocytes. PAI-1 mRNA, determined by RT-PCR, was identified in cultured cells (Fig. 1Go). From five separate cultures of human sc adipose tissue (all women; BMI, 19–42), mean PAI-1 antigen levels in the conditioned medium were 333 ± 64.8 ng/mL, with an average of 25,000 cells plated from each individual. PAI-1 was not detected in the medium alone. These experiments clearly indicate that human preadipocytes synthesize and secrete PAI-1.



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  		Figure 1. RT-PCR of PAI-1 mRNA from three separate human sc preadipocyte cultures. Human ß2-microglubulin (ß2M) mRNA amplified from the same samples is also shown. Lane 1, Female, 35 yr old, BMI of 22; lane 2, male, 28 yr old, BMI of 26; lane 3, male, 44 yr old, BMI of 30.

 
In smooth muscle cells, PAI-1 affects migration through regulating the interaction of {alpha}Vß3 integrin with the extracellular matrix component vitronectin, but whether human preadipocytes or adipocytes express specific cellular adhesion receptors or synthesize vitronectin is not known. Therefore, it was necessary to identify these molecules in adipose tissue before pursuing functional studies of PAI-1 in preadipocytes. Flow cytometric data (Fig. 2Go) from six separate human preadipocyte cultures representing both sexes and with various BMIs clearly indicate that human preadipocytes express the {alpha}Vß3 integrin, a novel finding with respect to adipose tissue physiology. The initial search for vitronectin in adipose tissue employed assessment of vitronectin mRNA from cultured preadipocytes and adipose tissue fractions using RT-PCR. As shown in Fig. 3Go, vitronectin mRNA was clearly identified in human liver, but not in adipose tissue fractions. Vitronectin mRNA was also undetected in preadipocytes (data not shown). A capture assay using a monoclonal antibody against human native vitronectin was therefore developed for identification and quantification of vitronectin in human adipose tissue. Using this technique, vitronectin was identified in significant quantity in human adipose tissue, but only in the stromal-vascular fraction (Table 1Go).



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  		Figure 2. Flow cytometric identification of {alpha}vß3 integrin in human preadipocytes. Human umbilical vein endothelial cells (HUVEC) were used as controls and are shown in the top panel. Each panel of preadipocytes represents a culture from a different individual, and various BMIs are represented.

 


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  		Figure 3. RT-PCR of vitronectin mRNA from human adipose tissue. Lane 1, Stromal-vascular fraction from the abdominal sc adipose tissue of a 48-yr-old woman with a BMI of 22; lane 3, whole tissue preparation from the abdominal sc depot of a 60-yr-old man with a BMI of 28; lane 5, adipocyte fraction from the breast sc adipose tissue of a 44-yr-old woman with a BMI of 37; lane 7, human liver. Lanes 2, 4, 6, and 8 are the no RT lanes for each corresponding sample. Vn, Vitronectin;. ß2M, ß2-microglubulin.

 

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  		Table 1. Vitronectin content of human adipose tissue and vessels

 
Having identified PAI-1, {alpha}Vß3 integrin, and vitronectin in components of human adipose tissue, two separate assays were performed to determine the effects of PAI-1 on preadipocyte attachment and migration. In the first assay, human preadipocytes from three separate cultures were preincubated with varying concentrations of PAI-1, followed by seeding onto a vitronectin-coated plate. PAI-1 inhibited attachment in a concentration-dependent manner, with an estimated IC50 of 13.3 nmol/L (Fig. 4Go). Latent PAI-1 (1 µmol/L) was ineffective in preventing attachment. These data suggest that PAI-1 in the local cellular environment can prevent a detached preadipocyte from reattaching to the extracellular matrix protein vitronectin. In a separate set of experiments, either active or latent PAI-1 (1 µmol/L) was added directly to the vitronectin-coated Transwell, then preadipocytes were seeded and assessed for capability to actively migrate. Again, these results (Figs. 5Go and 6Go) indicate that active PAI-1, but not latent PAI-1, reduces the migration of human preadipocytes. The results of these two separate studies are in agreement with those performed by others using smooth muscle cells (7) and indicate a regulatory role for PAI-1 in cell migration. This is the first time these components have been found in human preadipocytes.



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  		Figure 4. Dose-dependent effect of PAI-1 on preadipocyte attachment. Data represent the mean ± SEM of three separate experiments from three different cultures (a 61-yr-old man with a BMI of 34, a 26-yr-old woman with a BMI of 30, and a 35-yr-old woman with a BMI of 22).

 


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  		Figure 5. Effect of 1 µmol/L PAI-1 on migration of human preadipocytes through vitronectin-coated Transwells. *, P < 0.05 compared to serum (0.5%) and latent PAI-1 (1 µmol/L). Data were obtained from four individual cultures (two men and two women; age, 26–61 yr; BMI, 22–34).

 


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  		Figure 6. Representative images of the effect of PAI-1 on human preadipocyte migration. A solution of 1 µmol/L of either active or latent PAI-1 was added to a vitronectin-coated Transwell, followed by addition of cells suspended in 0.5% serum. Data from these experiments are quantitated in Fig. 5Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PAI-1 synthesis was originally investigated in a variety of tissues, including liver, spleen, kidney, and lung (19), with adipose tissue being initially identified as a potential site of PAI-1 production by Sawdey and Loskutoff (20). They examined a variety of tissues from mice using ribonuclease protection analysis, observing abundant PAI-1 mRNA expression in adipose tissue. More recently, PAI-1 mRNA has been identified in human adipose tissue explants (21, 22) and human adipose tissue primary cultures (23). Because of the relative mass that adipose tissue represents to total body weight, it has been hypothesized that adipose tissue is the main tissue source of the elevated plasma PAI-1 in obesity (24), contributing significantly to the cardiovascular pathologies of this disease.

Although experimental evidence suggests that human adipose tissue is a significant source of PAI-1, the physiological relevance of the synthesis of a fibrinolytic regulatory protein by adipose tissue remains an enigma. Because PAI-1 affects cell migration in smooth muscle, epithelial, and endothelial cells (7, 9, 25), we investigated the role of PAI-1 in the motility of the human adipocyte precursor cell, the preadipocyte. Our results clearly indicate that human preadipocytes exhibit the ability to migrate, and that this process is regulated by PAI-1. In addition, this cell type expresses additional components required for autocrine regulation of migration, including the membrane-associated {alpha}Vß3 integrin. The mediation by PAI-1 of the interaction between the membrane integrin and the matrix vitronectin is a high affinity process that is dependent upon the structural conformation of the serpin, as indicated by an IC50 in the low nanomolar range for active PAI-1, and the inactivity of latent PAI-1. As PAI-1 has been reported to reach local concentrations several thousand-fold greater than the IC50 (26), the effect on preadipocyte migration may have physiological relevance.

The final component required for PAI-1-mediated migration, vitronectin, is an adhesive extracellular matrix glycoprotein that binds PAI-1 with high affinity (27). The liver is the predominant organ for vitronectin synthesis, whereas the involvement of adipose tissue is controversial, as mRNA has been detected in murine adipose tissue by PCR, but not by Northern analysis (28). It is important to note, however, that vitronectin gene expression or protein identification has never been performed in human adipose tissue or its cellular components. Using PCR, we were unable to identify vitronectin mRNA in either cultured preadipocytes or adipocytes isolated from tissue biopsies, although we did observe gene expression in human liver. However, we identified significant quantities of vitronectin in homogenates of the stromal-vascular fraction of both visceral and sc adipose tissue. Using plasma (29) and vessel samples for comparison, these studies are the first to identify vitronectin in human adipose tissue, further localizing it to the stromal-vascular compartment. It is a reasonable assumption that vitronectin is transported to adipose tissue via the circulation, where it is bound by its receptor, {alpha}Vß3 integrin, and PAI-1.

In obesity, body fat mass increases through the enlargement of existing fat cells, but in gross obesity there is also an increase in cell number (12, 13). Classic studies on the development of human adipose tissue indicate that matrix deposition is followed by vascular growth within the matrix (15). Primitive fat organs, composed of mesenchymal cells, then localize and spread along established capillaries, eventually filling with lipid (15). Therefore, although preadipocyte migration is a prerequisite for adipose tissue development, the regulatory mechanisms have not been extensively studied. Our present findings indicate a functional role for PAI-1 in preadipocyte migration and expand upon previous studies from our laboratory that observed reduced preadipocyte PAI-1 synthesis once confluence was attained and active migration had ceased (23).

PAI-1 has been proposed to be involved in vascularizing murine tumors (8), and the vitronectin receptor, {alpha}Vß3 integrin, has been identified as important to angiogenesis, vascular remodeling, and developmental neovascularization (30), albeit in tissues other than adipose. PAI-1 can also affect cell surface proteolysis by inhibiting urokinase plasminogen activator (31), but whether this role exists in adipose tissue development has not been investigated. As adipose tissue is one of the most highly vascularized tissues (32), and adipogenesis proceeds along clearly established capillaries, the identification of molecules in adipose tissue that regulate cell migration and angiogenesis may be important for understanding the interaction of adipocytes with the stromal-vascular component of adipose tissue. Continued synthesis of PAI-1 by terminally differentiated adipocytes and the elevated plasma PAI-1 found in obese humans may therefore reflect the role of PAI-1 in adipose tissue development, which is triggered in adult obesity as a response to adipose tissue expansion. As the signals responsible for the early stages of adipogenesis remain largely unknown, the production by primordial fat cells of significant levels of a molecule capable of regulating migration, angiogenesis, and local proteolysis warrants further investigation.


   Acknowledgments
 
We appreciate the technical consultation of Dr. Daniel A. Lawrence (American Red Cross Holland Laboratory, Rockville, MD).

Received December 9, 1999.

Revised February 26, 2000.

Accepted March 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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I. Chowers, Y. Kim, R. H. Farkas, T. L. Gunatilaka, A. S. Hackam, P. A. Campochiaro, S. C. Finnemann, and D. J. Zack
Changes in Retinal Pigment Epithelial Gene Expression Induced by Rod Outer Segment Uptake
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S. Urs, C. Smith, B. Campbell, A. M. Saxton, J. Taylor, B. Zhang, J. Snoddy, B. Jones Voy, and N. Moustaid-Moussa
Gene Expression Profiling in Human Preadipocytes and Adipocytes by Microarray Analysis
J. Nutr., April 1, 2004; 134(4): 762 - 770.
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G. J. Hausman and R. L. Richardson
Adipose tissue angiogenesis
J Anim Sci, March 1, 2004; 82(3): 925 - 934.
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T. Yokota, C. S. R. Meka, T. Kouro, K. L. Medina, H. Igarashi, M. Takahashi, K. Oritani, T. Funahashi, Y. Tomiyama, Y. Matsuzawa, et al.
Adiponectin, a Fat Cell Product, Influences the Earliest Lymphocyte Precursors in Bone Marrow Cultures by Activation of the Cyclooxygenase-Prostaglandin Pathway in Stromal Cells
J. Immunol., November 15, 2003; 171(10): 5091 - 5099.
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