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Isolation and characterization of islet stellate cells in rat a

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Min Zha , Fengfei Li , Wei Xu , Bijun Chen & Zilin Sun a

Department of Endocrinology; Zhongda Hospital; Institute of Diabetes; Medical School; Southeast University; Nanjing, China Published online: 01 Apr 2014.

Click for updates To cite this article: Min Zha, Fengfei Li, Wei Xu, Bijun Chen & Zilin Sun (2014) Isolation and characterization of islet stellate cells in rat, Islets, 6:2, e28701, DOI: 10.4161/isl.28701 To link to this article: http://dx.doi.org/10.4161/isl.28701

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Research Paper

Research Paper

Islets 4, e28701; May 2014; © 2014 Landes Bioscience

Isolation and characterization of islet stellate cells in rat Min Zha, Fengfei Li, Wei Xu, Bijun Chen, and Zilin Sun* Department of Endocrinology; Zhongda Hospital; Institute of Diabetes; Medical School; Southeast University; Nanjing, China

Keywords: islet stellate cell, ISC, pancreatic stellate cell, PSC, islet

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Abbreviations: PSCs, pancreatic stellate cells, T2DM, type 2 diabetes mellitus; ECM, extracellular matrix; ISCs, islet stellate cells; α-SMA, α-smooth muscle actin; GFAP, glial fibrillary acidic protein; col-I, collagen types I; col-III, collagen types III; FN, fibronectin; CCK-8, cell counting kit-8; DTZ, dithizone; FBS, fetal bovine serum; ROS, reactive oxygen species; MMP, metalloproteinase; RAS, renin-angiotensin system; DMEM/F12, Dulbecco’s modified Eagle’s medium/Ham’s F12; PFA, paraformaldehyde

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs. PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-I, col-III and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.

Introduction The pathogenesis of fibrosis of the exocrine pancreas during the progression of chronic pancreatitis or in pancreatic cancer has been well studied and it is now clear that PSCs play a pivotal role in the fibrotic response. PSCs were first identified as fat-storing cells in the pancreas of vitamin A-loaded mice1 and were subsequently identified in normal rat and human pancreatic tissue2 from which they can be isolated and expanded by in vitro culture.3,4 Under normal conditions, PSCs are quiescent and are characterized by cytoplasmic lipid droplets rich in vitamin A. The PSCs are rapidly activated in response to pancreatic injury and inflammation or exposure to inflammatory cytokines or oxidant stress. Upon activation PSCs start to proliferate, change their morphology into myofibroblast-like cells, upregulate the expression of α-SMA, and secrete ECM components.5 Since the first identification of PSCs, numerous in vivo and in vitro studies have provided strong evidence of a central role of PSCs in pancreatic fibrogenesis associated with chronic pancreatitis and pancreatic cancer.5-12 Fibrotic responses to inflammatory insults may also be important in pathological responses in the endocrine portion of the pancreas, the islets of Langerhans. Islet fibrosis has been

demonstrated in the pancreas of humans with T2DM, and in a number of animal models of diabetes,13-18 where it is associated with the late stage of β cell failure during the progression to T2DM.19 However, the mechanisms responsible for the development of islet fibrosis are largely unknown. Previous reports localized PSCs to the inter-lobular and inter-acinar regions, but not in association with the islets.3,4 However, more recent studies have described α-SMA positive cells, identified as PSCs, in the islets of humans with T2DM and in animal models.16,20-22 In the current study we identify an α-SMA positive population of stellate cells within isolated islets which are similar, but not identical to classical PSCs, and which we have called islet stellate cells. In view of the central role of PSCs in pancreatic fibrosis, we suggest that ISCs may contribute to the islet fibrosis in the development of T2DM.

Results Isolation and culture of ISCs from isolated rat islets Islets were identified in the pancreatic digest after DTZ staining as red-stained clusters among the unstained exocrine digest. Islets were handpicked and incubated in RPMI1640

*Correspondence to: Zilin Sun; Email: [email protected] Submitted: 02/17/2014; Revised: 03/23/2014; Accepted: 03/29/2014; Published Online: 04/01/2014 http://dx.doi.org/10.4161/isl.28701 www.landesbioscience.com Islets e28701-1

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Figure 1. Isolation and culture of ISCs from isolated rat islets. Islets were characterized in the pancreatic digest after DTZ staining as red-stained clusters among the unstained exocrine digest (a). Isolated islets adhered to the bottom of culture dish, and acted as explant cultures from which there was a progressive outgrowth of proliferating cells from the edge of the islets (b: 0 h; c: 36 h; d: 48 h; e:72 h; f and g: 7 d). After 72 h in primary culture, we immune-stained the islet and the proliferating cells with antibodies against insulin (h: insulin; i: DAPI). Islet β-cells showed positive insulin immunoreactivity, but the proliferating adherent cell outgrowth did not express insulin.

containing 10% FBS. After 36 h islets adhered to the bottom of culture dish, and acted as explant cultures from which there was a progressive outgrowth of proliferating cells from the edge of the islets. These cells adhered to the culture dish and assumed a stellate, angular appearance without any detectable lipid droplets in the cytoplasm. These cells exhibited similar appearance to classical PSCs, but their rate of proliferation was slower than that described for classical PSCs. Within 7 d the proliferating, adherent stellate cells had formed a confluent monolayer of cells (Fig. 1). In order to exclude the possibility of β-cell out-growth, we immune-stained the islet and the proliferating cells with antibodies against insulin after 72 h in culture. As expected the islet β-cells showed positive insulin immunoreactivity, but the proliferating adherent cell outgrowth did not express insulin (Fig. 1). Activation of ISCs PSC activation is associated with the disappearance of lipid droplets from the cytoplasm and upregulation of the expression of α-SMA, so we compared these characteristics in classical PSCs and in our ISC populations, as shown in Figure 2. PSCs carried numerous prominent lipid droplets in the cytoplasm when they migrated from pancreas blocks after 48 h, which persisted unchanged for up to five days in culture. In contrast, after 48 h culture ISCs containing lipid droplets were very rarely observed, and all the lipid droplets were gone from the ISCs by

72 h (Fig. 2A). The activation of PSCs and ISCs was also assessed by measuring α-SMA expression by immunostaining, after 72 h outgrowth from primary pancreas blocks or isolated islets. Consistent with the maintained lipid droplets, PSCs were negative for α-SMA. In contrast, the ISCs showed almost 100% positive immunostaining, consistent with their being activated (Fig. 2B). Previous studies have shown PSCs are activated after one passage in tissue culture in vitro. In this study it was observed that both passaged PSCs and ISCs lost lipid droplets and expressed α-SMA, consistent with activation of both cell populations, with no obvious morphological differences between them (Fig. 3). Characterization of ISCs Immunofluorescence visualization of markers for PSCs (α-SMA, vimentin, GFAP) was used to characterize the ISCs at passage 3 (8 wk in culture). As shown in Figure 3, both populations of stellate cells (a-c, PSCs; d-f, ISCs) were positive for α-SMA, vimentin and GFAP. ECM synthesis by PSCs and ISCs A panel of antibodies directed against different ECM components was used to assess the synthesis of col-I, col-III, and FN by the stellate cell populations (passage 3). As shown in Figure 4, both PSCs and ISCs were immunopositive for col-I, col-III, and FN,

respectively. Proliferation and migration of stellate cell populations To assess the rate of cell proliferation of PSCs and ISCs (passage 3), we used the CCK-8 assay to measure the number of viable cells during a 72 h culture period after seeding 2 × 103 cells/ well in 96-well microplates, as shown in Figure 5. After 12 h the cells attached to the plate, and there was no significant difference between the absorbance measurements at A450nm in PSCs and ISCs groups, which indicated the number of PSCs and ISCs were similar at this time point. By 36 h in culture the number of PSCs was significantly greater than that of ISCs, and this difference was further extended after 72 h in culture, demonstrating that the ISCs proliferated at a significantly slower rate than the PSCs (Fig. 5). Migration was assessed by the wound-healing assay and the transwell migration assay, which are well-established in vitro systems for measuring cell motility.23,24 In wound healing assay, 24 h after the wound formation, the number of ISCs migrating into the cell-free area was significantly less than that of PSCs (33.8 ± 8.0% of control, P < 0.01, Figure 6A). In accordance with this, ISCs also showed less motility in the transwell migration assay. Thus, after 24 h the number of ISCs migrating through the membrane was significantly less than that of PSCs (56.8 ± 19.1% of control, P < 0.01, Figure 6B).

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Figure 2. Lipid droplets (A) and expression of α-SMA (B) in ISC and in classical PSCs populations were compared. (a-f: Pancreas block and the proliferating PSCs; g-l: Islet and the proliferating ISCs) (A) Lipid droplets in cytoplasm were detected by Oil red O staining. PSCs carried numerous lipid droplets when they migrated from pancreas blocks after 48 h (a and b), which persisted unchanged during 72 h in culture (c and d). After 72 h in culture, immunofluorescent staining for α-SMA showed PSCs were negative for α-SMA (e: α-SMA; f: DAPI). In contrast, after 48 h culture ISCs containing lipid droplets were very rarely observed (g and h), and all the lipid droplets were gone by 72 h (i and j), when ISCs were positive for α-SMA (k and j).

Discussion The presence of PSCs in the pancreas, and their role in fibrotic responses to injurious stimuli, is well established,11,25,26 but it remains to be established whether a similar cell population resides within the endocrine pancreatic islets. In the present study, we have used explant techniques to isolate stellate cell populations by outgrowth from small blocks of rat pancreas (PSCs),4 and from isolated rat islets (ISCs), and have compared their phenotypic characteristics. Neither cell population expressed insulin, so we can exclude the possibility of β-cell outgrowth. Classical PSCs are characterized by a range of markers including the presence of cytoplasmic lipid droplets and the expression of α-SMA, vimentin and GFAP.3,4 PSCs normally exist in the pancreas in a quiescent (non-activated) state, characterized by

numerous vitamin A containing lipid droplets in the cytoplasm. However, a range of injurious stimuli can switch PSCs from the quiescent state to an activated state. Thus, inflammatory cytokines, oxidant stress or other damage in acute or chronic pancreatitis or other pancreatic diseases can lead to a dynamic cascade of mechanisms beginning with acinar cell injury and necrosis and followed by inflammation, activation of macrophages, aggregation of platelets, release of growth factors and ROS, which subsequently change the phenotype of PSCs from the fatstoring, quiescent state to a highly active ECM-producing cell type. In addition, PSCs have the capacity to synthesize MMPs, including MMP-2, MMP-9, and MMP-13, and their inhibitors TIMP-1 and TIMP-2. In this way they maintain the balance between synthesis and degradation of ECM and may be responsible for the maintenance of normal ECM turnover and the normal architecture of the pancreas.27 In pathological environments

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the activated PSCs also reduce degradation of ECM. The consequent imbalance between the ECM synthesis and degradation results in a net matrix accumulation, eventually leading to development of pathological fibrosis which is associated with pancreatitis and pancreatic cancer.5-12,27 The ISCs which we have isolated in the current study share many phenotypical markers with classical PSCs, but are not an identical population to PSCs. The ISCs contained some lipid droplets in the cytoplasm at the initial stages of the outgrowth from islet but the droplets were significantly fewer than in the PSCs, and disappeared within 72 h culture in vitro, whereas PSCs maintained lipid Figure 3. Immunofluorescence visualization of markers for PSCs was used to characterize the droplets for many days in culture. In addition ISCs after passage 3. Immunofluorescent staining for α-SMA, GFAP, vimentin was performed the majority of ISCs were α-SMA-positive on PSCs (a-c) and ISCs (d-f) respectively. Both populations of stellate cells were positive for throughout the culture period whereas PSCs α-SMA (a and d), vimentin (b and e) and GFAP (c and f). did not express α-SMA. As the disappearance of lipid droplets and the expression of α-SMA are two established markers for the activation of PSCs, our results suggest that ISCs also exist in quiescent and activated states, and that ISCs are much more readily activated than PSCs in vitro. We also demonstrated that both populations of stellate cells were proliferative and highly motile in several different in vitro assays. However, we again noted differences between the two populations, with ISCs being significantly less proliferative and less motile than PSCs. The existence of a specialized stellate cell population within pancreatic islets may be of functional significance if the ISCs perform a similar role to PSCs. Islet fibrosis has been observed in the late stage of β-cell failure Figure  4. Immunofluorescence visualization of ECM components in PSCs and ISCs. Immunofluorescent staining for Col-I, Col-III and FN was performed on PSCs (a-c) and ISCs during the progression to T2DM in diabetic (d-f) respectively. Both PSCs and ISCs were immunopositive for col-I (a and d), col-III (b and e) patients as well as in animal models, with and FN (c and f), respectively. increased deposition of many ECM components, including Col-I, Col-III, and FN.1318 The precise mechanisms underlying islet fibrosis are largely unknown, although the deposition of islet amyloid, activation of RAS and oxidative stress have all been implicated.16,17,28-30 Our demonstration of a specialized ISC population in rodent islets is consistent with an important role for these cells in islet fibrosis, in an analogous manner to that of classical PSCs in pancreatic Figure  5. Proliferation of ISCs and PSCs in vitro. Cell proliferation was determined with the CCK-8 kit. By 36 h in culture the number of PSCs was significantly greater than that of ISCs, and this difference was further extended after 72 h in culture, demonstrating that the ISCs proliferated at a significantly slower rate than the PSCs. Data were expressed as mean ± SE (n = 15), ** P < 0.01, ISCs vs. PSCs. (The error bars were smaller than the symbols).

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Figure 6. Migration of ISCs and PSCs in vitro. (A) In wound healing assay, 24 h after the wound formation, the number of ISCs migrating into the cell-free area was significantly less than that of PSCs. Data were expressed as mean ± SE (n = 5), ** P < 0.01, ISCs vs. PSCs. (B) In transwell migration assay, cells adhering to the lower side of the insert membrane were purple after crystal violet staining. After 24 h the number of ISCs migrating through the membrane was significantly less than that of PSCs (n = 5), ** P < 0.01, ISCs vs. PSCs. The brown dots are pores in the membrane.

fibrosis.11,25,26 This proposal is supported by several recent studies suggesting that ISCs might play an important role in islet fibrosis in T2DM.16,20,21,29,30 For example, increased ISCs have been detected in pancreatic islets in patients with T2DM. Similarly, in several studies using different animal models of T2DM, treatments to suppress islet fibrosis and improve β-cell function were associated with significant reductions in the numbers of intraislet stellate cells.16,29 These studies provided compelling evidence for an association between activated ISCs and the development of islet fibrosis in T2DM. Moreover, similar cells were isolated from chick islets by S.P. Datar. It was reported that chick ISCs were positive for desmin, vimentin and fibronectin, which could be differentiated into islet-like clusters. But chick ISCs showed spontaneous adipogenesis after 2nd and 3rd passages, which was different in rat ISCs.22 The diabetic environment provides numerous signals which could lead to stellate cell activation. Thus, previous studies using classical PSCs have demonstrated that hyperglycemia,16,31 activation of the RAS,32 oxidative stress,17,30 platelet-derived growth factors,33 and inflammatory cytokines34 all enhance PSC activation and proliferation. The environment of the diabetic islet is characterized by the presence of the hyperglycemia 35-37

and oxidative stress,38-40 as well as macrophage infiltration and increased expression of inflammatory molecules such as IL-1, IL-6, TNF-α, MCP-1, and MIP-1α.14,35,36 Given the phenotypical similarities between ISCs and PSCs, it seems inevitable that the ISCs will be activated by the diabetic environment, with their subsequent proliferation and production of ECM components leading to the islet fibrosis.25 In conclusion, our study has provided evidence that islets contain stellate cells which exhibit features similar to those described for PSCs, but which are not identical to classical PSCs in terms of activation, proliferation, and motility. We propose that activation of these islet-derived stellate cells may be responsible for the islet fibrosis in T2DM.

Materials and Methods Animals Wistar rats were housed three per cage under a 12/12 h light/dark cycle and given access to food and water ad libitum. After a 1-wk acclimation period, the rats were used as pancreas donors after anesthesia.41 Housing and animal experiments

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were approved by Southeast University Animal Care and Use Committee according to institutional guidelines and national animal welfare. Isolation and culture of islets Islets were isolated from rat pancreas using collagenase V (1 mg/ml, Sigma), and handpicked under a stereomicroscope, as described previously.42 The islets can be characterized by DTZ staining (0.1g/l, Sigma).43 Freshly isolated islets were subsequently cultured in Corning culture dishes with 10ml DMEM/ F12 (1:1 v/v) with 10% FBS (Gibco) at 37 °C in a 5% CO2-air humidified atmosphere. Isolation and culture of PSCs PSCs were isolated from 8-wk-old wistar rats using explant techniques.4 Briefly, pancreas was isolated and cut into small tissue blocks (0.5–1mm3) which were seeded into uncoated culture dishes in the presence of 10% FBS in DMEM/F12. Tissue blocks were cultured at 37 °C in a 5% CO2-air humidified atmosphere. Oil red O staining Samples were fixed in 4% PFA in PBS for 20 min at room temperature. Oil red O (Sigma) staining was performed by incubating PFA-fixed material for 10 min a saturated solution of Oil red O in isopropanol, followed by washing with distilled water. Images were taken using a phase contrast microscopy (Zeiss). Immunostaining Cells were fixed in 4% PFA in PBS for 20 min at room temperature. Immunofluorescent staining of stellate cells for α-SMA, GFAP, vimentin, Col-I, Col-III and FN was performed. Cells were incubated overnight at 4 °C with primary antibody (Abcam, 1:200), followed by a 1 h treatment with secondary antibody (Jackson ImmunoResearch Laboratories, 1:100). The sections in negative control group were incubated with PBS, instead of primary antibody. And the results indicated that the antibody and the staining worked well. Morphometric analyses were performed using Image J software. CCK-8 assay Cell proliferation was determined with the CCK-8 kit (Keygen Biotech). Cells were suspended at a final concentration of 2 × 103 cells/well and cultured in 96-well microplates for 12, 36 and 72 h, after which CCK-8 reagent (10 μl) was added to each well containing 100 µl of culture medium and the plate was incubated for References 1.

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1 h at 37 °C. Viable cells were evaluated by A (A450nm) using auto microplate reader (Wellscan), such that A450nm was proportional to the rate of cell proliferation. All experiments were performed in quintuplicate on three separate occasions (n = 15). Cell migration assays The transwell migration assay was performed in 6-transwell plates using inserts with a pore size of 8 μm (Millipore). One × 106 stellate cells were serum-starved for 24 h, after which cells were cultured in serum-free medium in the inner compartment of the transwell insert, while the plate wells which received the inserts contained DMEM/F12 supplemented with 10% FBS. The cells were incubated for 48 h at 37 °C, then fixed with 2% glutaraldehyde and stained with 1% crystal violet for 10 min. The cells that had not migrated through the membrane were removed. Cells adhering to the lower side of the insert membrane were visualized by light microscopy and counted manually (n = 5). For the wound-healing assay, 1 × 103 cells were seeded in 24-well culture plates and grown to reach confluence. After serum starvation for 24 h, the monolayers were wounded by scraping off a strip of cells with a 10 μL pipette tip. After 24 h cells migrating into the wound boundaries were counted manually under a phase contrast microscope (n = 5). Statistical analysis Data were presented as the means ± SE. Statistical significance was determined by ANOVA or unpaired student’s t test, as appropriate, and differences between group were considered to be statistically significant when P < 0.05. All of the statistical analyses were performed using the Statistical Product and Services Solutions (SPSS) package (Version 11.5, SPSS Science). Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We are grateful to Professor Peter Jones (King’s College London) for his critical appraisal of this manuscript. We are grateful to Professor Sheng Zhao (Southeast University, China) for his technical assistance. The work was supported by the National Nature Science Foundation of China (30971399, 81370874 and 81170716).

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8. Bachem MG, Zhou Z, Zhou S, Siech M. Role of stellate cells in pancreatic fibrogenesis associated with acute and chronic pancreatitis. J Gastroenterol Hepatol 2006; 21(Suppl 3):S92-6; PMID:16958683; http://dx.doi.org/10.1111/j.1440-1746.2006.04592.x 9. Michalski CW, Gorbachevski A, Erkan M, Reiser C, Deucker S, Bergmann F, Giese T, Weigand M, Giese NA, Friess H, et al. Mononuclear cells modulate the activity of pancreatic stellate cells which in turn promote fibrosis and inflammation in chronic pancreatitis. J Transl Med 2007; 5:63; PMID:18053242; http://dx.doi.org/10.1186/1479-5876-5-63 10. Siech M, Zhou Z, Zhou S, Bair B, Alt A, Hamm S, Gross H, Mayer J, Beger HG, Tian X, et al. Stimulation of stellate cells by injured acinar cells: a model of acute pancreatitis induced by alcohol and fat (VLDL). Am J Physiol Gastrointest Liver Physiol 2009; 297:G1163-71; PMID:19779015; http:// dx.doi.org/10.1152/ajpgi.90468.2008

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www.landesbioscience.com Islets e28701-7

Isolation and characterization of islet stellate cells in rat.

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the l...
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