Adipose-derived mesenchymal stromal/stem cells in systemic sclerosis: Alterations in function and beneficial effect on lung fibrosis are regulated by caveolin-1

Abstract

The potential value of mesenchymal stromal/stem cell therapy in treating skin fibrosis in scleroderma (systemic sclerosis) and of the caveolin-1 scaffolding domain peptide in treating lung, skin, and heart fibrosis is known. To understand how these observations may relate to differences between mesenchymal stromal/stem cells from healthy subjects and subjects with fibrosis, we have characterized the fibrogenic and adipogenic potential of adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients, from mice with fibrotic lung and skin disease induced by systemic bleomycin treatment, and from healthy controls. Early passage systemic sclerosis adipose-derived mesenchymal stromal/stem cells have a profibrotic/anti-adipogenic phenotype compared to healthy adipose-derived mesenchymal stromal/stem cells (low caveolin-1, high α-smooth muscle actin, high HSP47, low pAKT, low capacity for adipogenic differentiation). This phenotype is mimicked by treating healthy adipose-derived mesenchymal stromal/stem cells with transforming growth factor beta or caveolin-1 small interfering RNA and is reversed in systemic sclerosis adipose-derived mesenchymal stromal/stem cells by treatment with caveolin-1 scaffolding domain peptide, but not scrambled caveolin-1 scaffolding domain peptide. Similar results were obtained with adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients and from bleomycin-treated mice, indicating the central role of caveolin-1 in mesenchymal stromal/stem cell differentiation in fibrotic disease.

Introduction

Previous studies have shown the potential value of adipose-derived mesenchymal stromal/stem cell (AT MSC) therapy in treating skin fibrosis in scleroderma (systemic sclerosis, SSc). However, there has been little work reported on treating lung fibrosis with AT MSCs or in combining AT MSCs with other therapies. MSCs are non-hematopoietic multipotent progenitor cells. MSCs were first isolated and identified from bone marrow (BM) aspirates ¹ and can also be isolated from adipose tissue and other sources. ² AT MSCs have gained attention because of their accessibility and ease of harvest. In comparison with other sources, AT MSCs are more easily cultured, grow more rapidly, can be cultured for longer times before becoming senescent, and in many cases are more effective in treating disease.^3,4 MSCs have three key features important in their isolation, characterization, and potential use in the treatment of diseases: (1) adherence to standard tissue culture plastic; (2) specific surface antigens: MSCs are CD73+, CD90+, CD105+, and CD45-; and (3) they can differentiate into many cell types including adipocytes, osteocytes, chondrocytes, fibroblasts, and myocytes. ² While MSC injection has beneficial effects on various diseases, it is unclear whether this is a direct effect (replacement of cell types) or an indirect effect (modulation of the tissue environment).

SSc is a chronic connective tissue disease involving the skin and internal organs. ⁵ Its principal pathophysiological manifestations are vasculopathy, autoimmunity, and extensive multi-organ fibrosis. The most devastating cases of SSc involve interstitial lung disease (ILD) which leads to progressive shortness of breath, poor quality of life, and then death within a few years. MSCs exhibit many functions relevant to SSc including immunomodulation, promotion of angiogenesis, and inhibition of fibrosis.^2,6 These pleiotropic properties make MSC-based therapy a promising innovation. Partially successful MSC therapy has been reported in a limited number of case studies. Beneficial effects were observed on digital ulcers. ⁷ The same team later reported four similar cases with improved limb cutaneous symptoms and no major adverse effects. ⁸ A patient treated with autologous BM MSCs had complete healing of acute gangrene in upper and lower limbs. ⁹ Injection into the affected skin of SSc patients of AT MSCs along with hyaluronic acid decreased skin thickness.^10–12 Despite these promising observations, one limitation to the widespread adoption of this approach is that the beneficial effect is of limited duration, so painful cell injections need to be repeated. In addition, to the best of our knowledge, there are no reports on the successful use of MSCs in treating lung fibrosis in SSc patients.

Caveolin-1 is a promising therapeutic target in fibrotic diseases. Caveolin-1 binds to and thereby inhibits the function or promotes the turnover of kinases in several signaling cascades.^13–19 It is underexpressed in several cell types in SSc patients and in animal models including fibroblasts and monocytes.^18,20–24 This deficiency leads to Col I overexpression by fibroblasts, monocyte hypermigration toward several chemokines, and to the enhanced differentiation of monocytes into CD45+/Col I+/α-smooth muscle actin+ (ASMA+) fibroblastic cells.^{18,22,24–26} The effects of caveolin-1 deficiency in cells and in animals can be reversed using the caveolin-1 scaffolding domain peptide (CSD).^27,28 CSD enters cells^29,30 and inhibits kinases similar to full-length caveolin-1.^31,32 In addition to the profibrotic effects of low caveolin-1 and their reversal by CSD in vitro, low caveolin-1 is profibrotic in vivo. Lung, skin, and heart fibrosis are observed in caveolin-1 knockout (KO) mice.^20,33–35 CSD also inhibits lung, skin, and heart fibrosis in animals.^25–27,36

While MSC-based therapy was proposed as a treatment for SSc because MSCs are immunomodulatory ² and SSc involves autoimmunity,^6,37,38 here we have addressed the possibility that AT MSC differentiation is altered in SSc. Consistent with the fact that increased transforming growth factor beta (TGFβ) signaling is observed in SSc MSCs, ³⁹ we found that SSc AT MSCs exhibit an enhanced profibrotic and inhibited adipogenic phenotype. Moreover, SSc AT MSCs, like SSc fibroblasts and monocytes, are deficient in caveolin-1. The fact that CSD reverses the altered phenotype and differentiation of SSc AT MSCs indicates that these alterations are regulated by caveolin-1. A similar profibrotic/anti-adipogenic phenotype was observed in AT MSCs from mice in which lung and skin fibrosis were induced using bleomycin.

Methods

Generating fibrosis in mice

The Pump Model ²³ is used to generate lung and skin fibrosis in mice. Briefly, under isoflurane anesthesia, mini-osmotic pumps (ALZET 1007D; DURECT Corporation, Cupertino, CA, USA) containing 100 µL of bleomycin (67 U/kg) or saline are implanted posterior to the scapulae of 8–10-week-old C57BL/6 mice. Pumps deliver 0.5 μL/h for 7 days and then are removed by day 10 per the manufacturer’s instructions.

Isolation of MSCs

Twenty one days after implantation of pumps containing saline or bleomycin into mice, inguinal fat is collected, minced, and digested with five volumes of 1 mg/mL collagenase (37°C, 30 min). The digest is diluted with maintenance medium (Dulbecco’s Modified Eagle’s Medium (DMEM)/10% fetal bovine serum (FBS)), filtered through a 70-µm mesh, and cells collected by centrifugation (800 g, 10 min, room temperature (RT)). Erythrocytes are lysed using ACK (ammonium-chloride-potassium) lysis buffer, cells collected by centrifugation, and cultured overnight in maintenance medium. Unattached cells are removed, and bound MSCs are further cultured for 1 week till near confluence.

Human MSCs were derived from abdominal fat tissue. SSc and SSc no ILD MSCs are a generous gift from Dr Del Papa (Milan, Italy). Healthy human MSCs are from Dr Del Papa and from Lonza. Human MSCs were maintained in the medium previously described. ⁴⁰ Early passage cells (passage 1 to 3) were used in all experiments except when late passage cells (passage 5) were specifically used in Figure 1(f).

Figure 1.

Baseline differences between healthy and SSc AT MSCs: AT MSCs from healthy subjects, SSc patients without ILD, and SSc ILD patients were cultured in maintenance medium for 10 days and then analyzed by IHC or western blotting. Throughout this figure, β-actin was the loading control for western blots. (a) Caveolin-1 levels were quantified by western blotting in extracts from four individuals in each group. Each symbol represents the data from one subject. p < 0.001 for SSc no ILD versus SSc ILD. (b) Healthy and SSc ILD AT MSCs were stained with the indicated antibodies and DAPI (nuclear stain). Representative results (n = 4) are shown. (c) Extracts of healthy and SSc ILD AT MSCs were analyzed by western blotting. Representative results are shown. (d) Western blot data from four experiments as in (c) were quantified densitometrically using ImageJ. The level of each protein in healthy AT MSCs was set to 100 arbitrary units. ***p < 0.001; **p < 0.01 for SSc ILD AT MSCs versus healthy AT MSCs. (e) Extracts of late passage healthy and SSc ILD AT MSCs were analyzed by western blotting. Representative results are shown. (f) Western blot data from four experiments as in (e) were quantified densitometrically using ImageJ. The level of each protein in healthy AT MSCs was set to 100 arbitrary units.

MSC differentiation and analysis

To determine whether human or mouse MSCs exhibited fibrogenic differentiation at baseline, cells were incubated for 10 days in maintenance medium and then analyzed by immunohistochemistry (IHC) and/or western blotting using appropriate antibodies. To induce fibrogenic differentiation in healthy human and mouse MSCs, cells were incubated for 10 days in maintenance medium supplemented with TGFβ (10 ng/mL). To determine the effects of knocking down caveolin-1 expression, healthy MSCs were treated with small interfering RNA (siRNA) targeting caveolin-1 according to the manufacturer’s instructions (Dharmacon, Lafayette, CO).

Adipogenic differentiation was induced by incubating cells for three cycles of 3 days in Adipocyte Induction Medium (Lonza, Walkersville, MD) plus 1 day in maintenance medium.Adipogenic differentiation was analyzed by IHC and/or western blotting using appropriate antibodies and by staining with an Oil Red O kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions.

Western blotting

MSC cultures were rinsed with phosphate-buffered saline (PBS). Lysis buffer (20 mM Tris-HCl (pH 7.5)/1% NP-40/100 mM NaCl/5 mM ethylenediaminetetraacetic acid (EDTA)/2 mM KCl) supplemented with 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture set V (Calbiochem, San Diego, CA, USA), and phosphatase inhibitors (10 mM sodium pyrophosphate, 5 mM NaF, 10 mM β-glycerophosphate, and 10 mM sodium orthovanadate) was added to the plates; the cells were scraped into the buffer, frozen, thawed; and the extract was clarified by centrifugation (10 min, 12,000 r/min, RT). The protein content of the supernatants was determined, sample buffer added, and the samples were boiled. Protein (5–40 µg) was loaded per lane depending on the avidity of the primary antibody to be used for detection.

Statistical analyses

Student’s t test is used to analyze data comparing two samples. When comparing three or more groups, two-way analysis of variance (ANOVA) is used with the Bonferroni post-test. In experiments involving western blots, immunoreactive bands were quantified by densitometry using ImageJ 1.32 NIH software. Raw densitometric data are analyzed using Prism 3.0 (GraphPad Software Inc., San Diego, CA, USA). Results are regarded as statistically significant if p < 0.05.

Results

Baseline differences between healthy and SSc AT MSCs

To determine the potential contribution of AT MSCs to the altered fibrogenesis and adipogenesis observed in SSc, several strains of AT MSCs from healthy subjects and SSc patients were compared as well as the role of caveolin-1 in regulating MSC differentiation. We first examined caveolin-1 levels at early passage and observed that while MSCs from SSc ILD patients expressed significantly decreased levels of caveolin-1, MSCs from SSc patients without ILD exhibited intermediate levels (Figure 1(a)). We further examined SSc ILD AT MSCs by IHC and western blotting and observed again that they are deficient in caveolin-1, overexpress the myofibroblast marker ASMA and the collagen chaperone HSP47, and exhibit decreased AKT activation (Figure 1(b)–(d)). The caveolin-1 deficiency is similar to what has been observed in fibroblasts and monocytes in SSc patients and linked to profibrotic behavior.^18,24

Although MSCs have been reported to be stable in phenotype in culture, at later passage, we did not observe these differences in caveolin-1, ASMA, HSP47, and AKT activation between healthy and SSc ILD AT MSCs (Figure 1(e) and (f)), suggesting that the phenotype of SSc AT MSCs is altered in vivo due to the profibrotic environment, and when cultured in vitro, they revert to a similar phenotype to healthy MSCs. To test this concept, healthy MSCs were treated in vitro with TGFβ. As predicted, this treatment enhanced ASMA expression (Figure 2(a)). To further implicate caveolin-1 in the fibrogenic behavior of AT MSCs, we used siRNA to decrease caveolin-1 levels and CSD to serve as a caveolin-1 surrogate. Indeed, in support of the profibrotic role of caveolin-1 depletion, specific siRNA decreased caveolin-1 levels and increased ASMA, HSP47, and pERK levels while having no effect on pAKT (Figure 2(b) and (c)). CSD inhibited both the baseline expression of ASMA in healthy MSCs and the enhanced expression observed in cells treated with TGFβ (Figure 2(a)), while the scrambled control version of CSD did not differ from vehicle in its effect.

Figure 2.

TGFβ and caveolin-1 siRNA treatment of healthy AT MSCs: treatments were performed as described in the “Methods” section. (a) MSCs were treated with CSD, scrambled CSD (Scr), and TGFβ as indicated. Representative IHC images of ASMA staining are shown. Staining was quantified densitometrically in arbitrary units using ImageJ (n = 4). The quantification is shown below the images. ***p < 0.001 for TGFβ versus control; ^^^p < 0.001 for TGFβ + CSD versus TGFβ alone. (b) Extracts of healthy AT MSCs treated with or without caveolin-1 siRNA were analyzed by western blotting with the indicated antibodies. Representative results are shown. (c) Western blot data from four experiments as in (b) were quantified densitometrically using ImageJ. The level of each protein in control healthy AT MSCs was set to 100 arbitrary units. ***p < 0.001; *p < 0.05 for caveolin-1 siRNA-treated MSCs versus control MSCs.

Altered induction of adipogenesis in SSc AT MSCs

The differentiation of healthy AT MSCs into adipocytes is widely studied. We find that with induction levels of the adipocyte markers FABP4 and PPARγ increase as does AKT activation which is known to be associated with adipogenesis ⁴¹ (Figure 3(a) and (b)). Similarly, caveolin-1 levels increase, in accordance with our previous results ²² positively linking caveolin-1 levels and function to adipogenesis. In contrast, levels of myofibroblast markers ASMA and Col I decrease when adipogenesis is induced (Figure 3(a) and (b)). When we examined the differentiation of SSc AT MSCs into adipocytes, in contrast to healthy AT MSCs, SSc AT MSCs responded poorly to induction, in that few cells expressed FABP4 and the majority expressed ASMA (Figure 4(a)–(d)). To demonstrate the role of caveolin-1 in the altered differentiation of SSc AT MSCs, the cells were treated with CSD during induction. As predicted, this treatment increased the number of FABP4+ cells and decreased the number of ASMA+ cells (Figure 4(a)–(d)), while the scrambled control peptide had no effect. Although healthy and SSc AT MSCs differed in FABP4 expression in response to induction, they did not differ in lipid droplet accumulation (Figure 4(e)).

Figure 3.

TGFβ and adipocyte induction of healthy AT MSCs: treatments were performed as described in the “Methods” section. (a) Extracts of healthy AT MSCs receiving the indicated treatments (±Induction ± TGFβ) were analyzed by western blotting with the indicated antibodies. Representative results are shown. (b) Western blot data from three experiments as in (a) were quantified densitometrically using ImageJ. The level of each protein in uninduced cultures without TGFβ was set to 100 arbitrary units except for FABP4 and PPARγ for which the level in induced cultures without TGFβ was set to 100 arbitrary units. ***p < 0.001; **p < 0.01 for uninduced + TGFβ versus uninduced alone and induced + TGFβ versus induced alone; ^^^p < 0.001 for induced alone versus uninduced alone.

Figure 4.

Altered adipocyte differentiation of SSc AT MSCs: healthy and SSc AT MSCs were induced for adipocyte differentiation ± CSD and the scrambled control peptide (Scr) as described in the “Methods” section. (a) ASMA levels in the indicated cultures were evaluated by IHC. Representative results are shown. (b) ASMA levels from three experiments as in (a) were quantified densitometrically using ImageJ. (c) FABP4 levels in the indicated cultures were evaluated by IHC. Representative results are shown. (d) FABP4 levels from three experiments as in (c) were quantified densitometrically using ImageJ. (e) Representative results from Oil Red O–stained induced cultures of healthy and SSc AT MSCs are shown. ***p < 0.001; *p < 0.05 for healthy + CSD versus healthy and SSc + CSD versus SSc. ^^^p < 0.001 for SSc versus healthy.

To model the onset of SSc, we differentiated healthy AT MSCs into adipocytes and then treated the cells with TGFβ. Even with this delayed treatment, TGFβ reversed adipogenesis (FABP4 expression) and promoted fibrogenesis (ASMA, Col I expression) (Figure 5(a) and (b)). Consistent with the positive relationship between adipogenic potential and AKT activation (Figures 1 and 3), TGFβ inhibited AKT activation (Figure 5(c) and (d)), thus blocking adipogenesis and altering kinase activation (Figure 5(a)–(d)). TGFβ also enhanced p38 activation (Figure 5(c) and (d)). CSD reversed the fibrogenic effect of TGFβ (increased ASMA and Col I expression) but did not restore FABP4 expression (Figure 5(a) and (b)). Neither TGFβ nor CSD affected PPARγ or caveolin-1 expression (Figure 5(a) and (b)). In contrast to effects of TGFβ on signaling, CSD inhibited p38 activation and restored AKT activation (Figure 5(c) and (d)). Thus, TGFβ inhibits the adipogenesis of AT MSCs and this effect is reversed by CSD both during induction and when AT MSCs are already differentiated into adipocytes.

Figure 5.

Effects of TGFβ and CSD on healthy MSCs already having undergone adipocyte differentiation: healthy AT MSCs were induced to differentiate into adipocytes and then treated ± TGFβ ± CSD as described in the “Methods” section. (a, c) Extracts of the indicated cultures were analyzed by western blotting with the indicated antibodies. (b, d) Western blot data from three experiments as in (a) and (c), respectively, were quantified densitometrically using ImageJ. The level of each protein in cultures that did not receive TGFβ or CSD was set to 100 arbitrary units. ***p < 0.001; **p < 0.01 for CSD versus control and CSD + TGFβ versus TGFβ alone. ^^^p < 0.001; ^^p < 0.01; ^p < 0.05 for TGFβ alone versus control alone.

AT MSCs in murine fibrosis induced by bleomycin

We routinely model SSc in mice by treatment with bleomycin delivered using subcutaneously implanted osmotic minipumps. ²³ Besides inducing lung fibrosis, this treatment results in the thickening of the dermis and the thinning of the intradermal adipose layer (similar to what is observed in SSc patients ⁴² ). Here, we have isolated AT MSCs from bleomycin- and saline vehicle-treated mice and have analyzed these cells using the same approaches described above for human AT MSCs. Similar to the human data, AT MSCs from bleomycin-treated mice contain much less caveolin-1 and much more ASMA, HSP47, and Col I than AT MSCs from saline-treated mice (Figure 6(a) and (b)). When AT MSCs were induced for adipogenesis, FABP4, PPARγ, and caveolin-1 were present at high levels in saline AT MSCs, but not in bleomycin AT MSCs (Figure 6(c) and (d)). Conversely, ASMA and Col I were present at high levels in bleomycin AT MSCs, but not in saline AT MSCs. Thus, the behavior of AT MSCs from bleomycin-treated mice is very similar to the behavior of AT MSCs from SSc patients or AT MSCs from healthy subjects that are treated with TGFβ after isolation.

Figure 6.

Differences between AT MSCs from healthy and bleomycin-treated mice. (a, b) AT MSCs isolated from saline-treated (healthy) mice and bleomycin-treated mice were cultured in maintenance medium for 10 days and then analyzed by IHC or western blotting. β-actin was the loading control for western blots. (a) Healthy and bleomcyin AT MSCs were stained with the indicated antibodies and DAPI (nuclear stain). Representative results (n = 3) are shown. (b) (Left) Extracts of healthy and bleomycin AT MSCs were analyzed by western blotting. Representative results are shown. (Right) Western blot data from three experiments were quantified densitometrically using ImageJ. The level of each protein in healthy AT MSCs was set to 100 arbitrary units. *p < 0.05 for bleomycin versus healthy AT MSCs. (c, d) Healthy (saline) and bleomycin AT MSCs were induced for adipocyte differentiation as described in the “Methods” section and then analyzed by IHC. (c) Saline and bleomcyin AT MSCs induced for adipocyte differentiation were stained with the indicated antibodies and DAPI (nuclear stain). Representative results are shown. (d) Levels of the indicated proteins in three experiments as in (c) were quantified densitometrically using ImageJ. ***p < 0.001; **p < 0.01 for bleomycin versus saline AT MSCs induced for adipocyte differentiation.

Discussion

The novel findings described here include the following: (1) SSc AT MSCs exhibit a profibrotic phenotype at baseline, in that they express low levels of caveolin-1 and high levels of ASMA and HSP47. A profibrotic phenotype is also generated in healthy AT MSCs by TGFβ treatment (which decreases caveolin-1 levels) or caveolin-1 siRNA treatment.

This profibrotic phenotype is reversed by CSD. Together, these observations indicate that the profibrotic phenotype of SSc AT MSCs is due to their deficiency in caveolin-1. AT MSCs from bleomycin-treated mice also exhibit a profibrotic phenotype. (2) SSc AT MSCs also exhibit an anti-adipogenic phenotype, in that they do not respond well to the induction of adipogenesis. Not only is their expression of adipogenic marker FABP4 inhibited, but also they continue to express high levels of the myofibroblast marker ASMA. CSD reverses the anti-adipogenic phenotype. The anti-adipogenic phenotype is also exhibited by AT MSCs from bleomycin-treated mice. (3) The profibrotic, anti-adipogenic phenotype of SSc AT MSCs is lost during prolonged culture. (4) AKT signaling may play a key role in the inhibition of adipogenesis observed in SSc AT MSCs. SSc AT MSCs are deficient in pAKT; both inhibited adipogenesis and loss of pAKT are induced by TGFβ treatment of healthy MSCs, differentiated adipocytes contain high levels of pAKT, and the loss of pAKT induced by TGFβ is reversed by CSD. These points are discussed below.

In general, previous studies have not revealed the wide range of differences between SSc and healthy AT MSCs that we report here. ⁴⁰ Previous studies were focused on MSC markers and proliferation rate, while this study is focused on differentiation and signaling. We agree with previous studies that the adipocyte differentiation of SSc and healthy MSCs is similar in terms of Oil Red O staining; however, they are different in terms of the expression of FABP4, a marker for mature adipocytes. ⁴³ The idea that FABP4 should be used rather Oil Red O to identify adipocytes is further supported by the fact that adipocytes are not the only cell type that can be Oil Red O+ (lipofibroblasts, ⁴⁴ foam cells ⁴⁵ ). While the functional similarity of AT MSCs and BM MSCs remains to be elucidated, our observation of low caveolin-1 level in SSc AT MSCs is similar to the observation that SSc BM MSCs are low in caveolin-1.^46,47 This may be related to the observation that SSc AT MSCs undergo senescence at early passage compared to healthy AT MSC, ⁴⁸ in that we find that early passage SSc AT MSCs exhibit low caveolin-1 levels which increase with passage. Thus, the increase in caveolin-1 in SSc AT MSCs may be associated with senescence, in agreement with observations in several cell types that an increase in caveolin-1 results in cell senescence. ⁴⁶ Further studies will be needed to explain why the constant, high level of caveolin-1 in healthy AT MSCs does not result in senescence.

We demonstrated previously that several cell types (fibroblasts, monocytes neutrophils, T cells) isolated from SSc donors were deficient in caveolin-1 leading to altered behavior and signaling.^18,24 Here, we show that AT MSCs are yet another cell type with low caveolin-1 and enhanced fibrogenic features when isolated from SSc ILD patients or from mice treated with bleomycin. Interestingly, SSc patients without ILD exhibit only a marginal decrease in caveolin-1 levels. The central role of caveolin-1 in fibrosis was demonstrated, in that some of the effects of low caveolin-1 occurred whether caveolin-1 levels were reduced using siRNA or by treatment with TGFβ and were reversed when cells were treated with the caveolin-1 surrogate CSD. These profibrotic effects included increased expression of Col I, HSP47, and ASMA and inhibition of adipogenic differentiation including decreased expression of adipogenic markers FABP4 and PPARγ. Moreover, when SSc AT MSCs are treated under conditions that induce adipogenesis in healthy AT MSCs, the SSc AT MSCs continue to express fibrogenic markers at high levels.

AKT is a key signaling molecule in these effects. pAKT is present at low levels in SSc AT MSCs. Induction of adipogenesis in healthy AT MSCs results in a large increase in pAKT. Treatment of these cells with TGFβ inhibits adipogenesis and concomitantly inhibits AKT activation. These effects were reversed by CSD. AKT activation has previously been found to positively regulate adipogenic differentiation based on the observations that insulin-like growth factor–binding protein 2 (IGFBP2) induces adipogenic differentiation of MSCs and increases AKT activation, that inhibitor-mediated blockage of AKT signaling dramatically reduces IGFBP2-mediated adipogenic differentiation, and that adipogenesis is inhibited in AKT KO mice.^41,49 These data are in agreement with our findings on a deficit in AKT activation in SSc AT MSCs and their decreased ability to differentiate into adipocytes.

The link between caveolin-1 and adipogenesis is also supported by genetic studies in mice and humans. Caveolin-1 KO mice exhibit defective adipose tissue lipid storage and produce only small adipocytes with lipid droplets of a reduced size. Caveolin-1 was recently identified as a locus for human congenital lipodystrophy, ¹³ indicating that caveolin-1 is also required for appropriate adipose lipid storage in humans. ⁵⁰

Given the demonstrated value of MSC therapy in treating skin fibrosis in SSc and of CSD in treating lung, skin, and heart fibrosis in animal models, the results of this study raise the interesting possibility of a synergistic effect of combined MSC/CSD therapy in fibrotic diseases. Experiments are now underway to test this possibility.