Thrombospondin 1 in hypoxia-conditioned media blocks the growth of human microvascular endothelial cells and is increased in systemic sclerosis tissues
© Morgan-Rowe et al; licensee BioMed Central Ltd. 2011
Received: 11 October 2010
Accepted: 2 June 2011
Published: 2 June 2011
Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. Vasculopathy is an early feature of the disease which leads to a chronic hypoxic environment in the tissues. Paradoxically, there is a lack of angiogenesis. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. We considered thrombospondin 1 (TSP-1) as a candidate antiangiogenic factor.
Conditioned media from human microvascular endothelial cells cultured in both normoxic and hypoxic environments were able to block endothelial cell proliferation, with the latter environment having a more profound effect. Filtration to remove > 100-kDa proteins or heparin-binding proteins from the conditioned media eliminated their antiproliferative effect. TSP-1 was expressed in high concentrations in the hypoxic media, as was vascular endothelial growth factor (VEGF). Depletion of TSP-1 from the media by immunoprecipitation reduced the antiproliferative effect. We then show that, in a dose-dependent fashion, recombinant TSP-1 blocks the proliferation of endothelial cells. Immunohistochemistry of skin biopsy material revealed that TSP-1 expression was significantly higher throughout the skin of patients with SSc compared with healthy controls.
Despite the environment of chronic tissue hypoxia in SSc, there is a paradoxical absence of angiogenesis. This is thought to be due in part to aberrant expression of antiangiogenic factors, including TSP-1. We have demonstrated that TSP-1 is released in high concentrations by hypoxic endothelial cells. The conditioned media from these cells is able to block proliferation and induce apoptosis in microvascular endothelial cells, an effect that is reduced when TSP-1 is immunoprecipitated out. Further, we have shown that recombinant TSP-1 is able to block proliferation and induce apoptosis at concentrations consistent with those found in the plasma of patients with SSc and that its effect occurs in the presence of elevated VEGF levels. Taken together, these data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues.
Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations . The pathogenesis of SSc has yet to be fully elucidated. Vasculopathy occurs early in the disease and precedes fibrosis . This is characterised by both abnormal vascular tone and endothelial cell damage . Endothelial apoptosis is seen in recent-onset SSc patients' dermal biopsies . Anti-endothelial cell antibodies have been detected in some but not all patients with SSc . These antibodies are capable of upregulating the expression of endothelial cell adhesion molecules and inducing apoptosis .
Other sources of early endothelial cell damage include reactive oxygen species, markers of which are found in the serum and urine of patients with SSc at higher levels than in controls [7, 8]. In addition, nitric oxide synthesis by endothelial cells is dysregulated in SSc because of suppression of the endogenous nitric oxide synthetase and upregulation of the inducible isoform. Because of this, proteins and lipids can become damaged in SSc by oxidation or nitrosylation .
Vascular damage clinically manifests in early SSc as Raynaud's phenomenon, sometimes seen many years before the other features develop . In addition, nailfold capillaroscopy is abnormal in early SSc, reflecting the endothelial vascular damage. The characteristic features of nailfold capillaroscopy in SSc include the presence of abnormal tortuous microvascular loops, the progressive loss of capillary density and areas of microvascular loss. Despite the chronic hypoxic environment and increased levels of proangiogenic vascular endothelial growth factor (VEGF) and its receptors, there is a failure of endothelial repair and an absence of angiogenesis in SSc [11, 12].
It has been postulated that the failure of angiogenesis in SSc may be due to a nonphysiological, overriding influence of antiangiogenic factors, which are present in high concentrations in the sera of patients with SSc [13–15]. Furthermore, plasma from patients with SSc has been shown to inhibit the migration and proliferation of microvascular endothelial cells .
Because of this, we became interested in the idea that hypoxic tissues are capable of releasing factors which interfere with reparative angiogenesis. We have gone on to test whether media from human cells cultured under hypoxic conditions can interfere with the growth of endothelial cells and whether the candidate antiangiogenic factor thrombospondin 1 (TSP-1) is induced in hypoxic cells or in the involved tissues of SSc patients.
The extended lifespan simian virus 40 transfected dermal human microvascular endothelial cell 1 (HMEC-1) cell line was employed. This line was generated by Ades et al.  and has been shown to maintain the phenotypic characteristics of small-vessel endothelial cells. For experiments, HMEC-1 cells were cultured in DMEM (Autogen Bioclear UK Ltd, Caine, Wiltshire, UK) with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin.
Cells were examined regularly by using phase contrast microscopy. Preparations which had reached confluence were trypsinised for secondary culture. Preparations which appeared to be infected by bacteria or failed to reach confluence after five days were discarded. Cells were passaged at confluence using 10% trypsin and split into equal thirds. The C2C12 mouse myoblast line 19 was also used. C2C12 cells were cultured on 0.01% gelatine plates in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin.
Cells were cultured in a custom-made hypoxic chamber placed within a tissue culture incubator and maintained at 37°C. The cells were plated onto 100-mm tissue culture plates and placed within the chamber, which had been primed for one hour by flushing with 4% CO2, 1% O2 and 95% N2. The chamber was then sealed, and hypoxic conditions were maintained for a further 24 hours prior to removal of the media for assay. Media were removed at the end of 24 hours of hypoxic culture for gas analysis and were found to have the following values (means ± SEM): pH 7.41 ± 0.04, pO2 6.4 ± 0.8 kPa and pCO2 5.2 ± 1.2 kPa. By comparison, media following culture under standard normoxic conditions for 24 hours had the following values (means ± SEM): pH 7.42 ± 0.04, pO2 16.2 ± 1.1 kPa and pCO2 4.8 ± 1.6 kPa.
Endothelial cell proliferation
HMEC-1 cells were harvested at 80% confluence and plated at 5 × 104 cells/well in 12-well plates, cultured for 24 hours and then cultured further in the presence or absence of 10% FCS with or without conditioned media from endothelial cells or skeletal muscle cells to test for antiproliferative effects of the conditioned media. After 24 hours of incubation, the cells were harvested by trypsinisation and counted in a haemocytometer. Each experiment was performed in triplicate.
In further experiments, HMEC-1 cells were cultured on 96-well plates with or without conditioned media from endothelial cells or with various concentrations of recombinant TSP-1 (R&D Systems Minneapolis, MN, USA). The number of viable cells was measured by WST-1 assay. In this assay, a tetrazolium dye undergoes a colour change depending on the presence of viable mitochondria. Ten microlitres of WST-1 were added to each well, and the cells were incubated for a further two hours. The colour intensity was read at 450 nm (reference wavelength 655 nm) in an ELISA plate reader.
Assay for TSP-1 and VEGF
Media were removed and assayed for TSP-1 and VEGF by ELISA (R&D Systems Pharmacia Biotech, Little Chalfont, UK). Experiments were performed in triplicate, and samples were assayed in duplicate according to the manufacturer's instructions. Standard curves were included in each assay plate.
Western blot analysis
Western lysate samples of 20 μL containing 10 μg of protein were run upon a ready-cast 4% to 12% Tris-Glycine gel (Novex. Ontario, Canada) alongside a broad-range protein marker (New England Biotech, Wakefield, MA, USA) at 125 V until the dye front had reached the bottom of the gel (approximately 1.5 hours) in Tris-Glycine Running Buffer (Invitrogen, Carlsbad, CA, USA). Proteins were electrophoretically transferred to nitrocellulose Hybond-C (Amersham Pharmacia, Little ChalfontBuckinghamshire, UK). Each membrane was briefly washed, and then nonspecific protein binding was blocked by one-hour incubation with 10% milk protein in PBS. Membranes were probed with antibodies against caspase 3 and cleaved caspase 3 (#9662 and #9661S; New England Biolabs, Wakefield, MA, USA), and binding was detected using labelled species-specific secondary antibody followed by avidin-biotin complex detection assay (Amersham Pharmacia).
Quantitative RT-PCR analysis
Primers used for qRT-PCR
For immunofluorescence, sections were permeabilised by washing three times in cold PBS containing 0.3% Triton X-100 at room temperature for 5 minutes, then washing them further in PBS containing 0.1% Tween. Nonspecific binding sides were blocked with 3% BSA and 10% serum in PBS with Tween for 30 minutes. Sections were immunostained with rabbit polyclonal anti-TSP-1 antibody (#sc-14013; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. As a negative control, samples were incubated with polyclonal rabbit immunoglobulin G (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with Alexa Fluor 568- or Alexa Fluor 594-labelled anti-rabbit antibody (Molecular Probes/Invitrogen, Eugene, OR, USA) (1:1000) for one hour. Nuclei were stained for 4',6-diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA) for 10 minutes. Glass coverslips were mounted onto slides with Vector aqueous anti-fade VECTASHIELD fluorescence mounting medium (Vector Laboratories) and sealed with nail polish. Sections were imaged with an Zeiss Axioplan microscope (Carl Zeiss, Heidenheim, Germany) at ×40 original magnification.
Conditioned media from hypoxic endothelial cells block proliferation
Antiproliferative effect of hypoxia-conditioned media is due to a heparin-binding macromolecule
TSP-1 is induced in hypoxia-conditioned media and blocks proliferation of HMEC-1 cells
In addition, TSP-1 mRNA levels in HMEC-1 cells cultured under normoxic and hypoxic conditions were assayed by qRT-PCR. Culture under hypoxic conditions for 24 hours led to the induction of TSP-1 expression (mean ± SEM normoxic cell TSP-1 copy number 3.19 ± 0.41, mean ± SEM hypoxic culture copy number 8.98 ± 2.39; P < 0.0035) (Figure 3).
TSP-1 is expressed by endothelial cells in normal skin and is induced in SSc-involved tissues
Also, we wanted to test whether free TSP-1 was present in the dermal interstitial fluid sampled from SSc and healthy control skin. In fact, in dermal interstitial fluids, TSP-1 was present only at very low levels and appeared absent from SSc dermal fluid (data not shown). We concluded that while TSP-1 was abundant within the dermis and epidermis in SSc, it was present only at low levels as a free, non-matrix-associated factor in the extracellular fluid.
Hallmarks of SSc are tissue ischaemia due to vascular injury, remodelling and stenosis; increased vascular tone; and microvascular damage. Despite the endothelial cell damage and activation and the ensuing chronic hypoxic environment, there is a failure of angiogenesis in SSc. It has been hypothesised that this might be due in part to an aberrant expression of antiangiogenic factors, and we have sought to investigate further the antiangiogenic effects of TSP-1 in the disease.
TSP-1 is derived from a large trimeric glycoprotein and functions as an adhesive matricellular factor modifying the binding of a variety of cells to extracellular matrix proteins [18, 19]. TSP-1 has multiple binding sites, including an amino terminal heparin-binding domain, a procollagen domain, epidermal growth factor-like repeats and an RGD (arginine-glycine-aspartic acid) integrin-binding region . The binding of TSP-1 to fibronectin, fibrinogen, laminin and collagen 5 has been described [21, 22]. TSP-1 is released from granules of platelets, from endothelial cells and from other cells, including keratinocytes and fibroblasts [23, 24]. Diverse functions of TSP-1 have been defined, including the adhesion of platelets to thrombin-containing clots, targeting of cells for apoptosis and clearance of apoptotic cells by macrophages . Studies of TSP-1-knockout mice have demonstrated that wounds heal more slowly and irregularly because of prolonged inflammation, delayed closure, scab loss and reepithelialisation. In hindlimb ischaemia in the TSP-1-knockout mice, enhanced angiogenesis and improved blood flow were shown [26, 27].
We were interested to find that when media from microvascular human endothelial cells cultured in hypoxia were added to proliferating endothelial cells, proliferation was blocked and apoptosis was induced. These effects were not observed when media from hypoxic muscle cells were used. This suggests the presence of a factor released by the hypoxic endothelial cells which was blocking proliferation. This effect was reduced by diluting the conditioning media, removing proteins that bind heparin and filtering large molecular weight proteins. This led us to consider whether TSP-1, a large glycoprotein with a heparin-binding domain, was a component of the culture media contributing to its antiproliferative and apoptotic effect. In keeping with this idea, we found that the concentration of TSP-1 was increased in media from hypoxic endothelial cells compared with normoxic endothelial cells and that TSP-1 mRNA levels were induced by hypoxic culture. Also, depletion of TSP-1 by immunoprecipitation partially blocked the inhibition of proliferation. These data are consistent with those in previous studies showing that TSP-1 can induce apoptosis of cells and that TSP-1 is hypoxia-inducible under some culture conditions [28, 29]. Furthermore, when we directly treated proliferating endothelial cells with recombinant TSP-1, there was suppression of proliferation and induction of apoptosis.
We also have shown that there is more TSP-1 expressed in the dermis and epidermis of patients with SSc than in healthy controls, with staining in keeping with the presence of TSP-1 in keratinocytes and dermal connective tissue. These results are consistent with published data showing that TSP-1 is elevated in SSc . Interestingly, the plasma levels of TSP-1 seen in SSc (mean ± SEM TSP-1 27.2 ± 8.5 ng/mL) are close to the levels that we found blocked endothelial cell proliferation and induced apoptosis in cultured endothelial cells.
One interesting result is that despite the induction of VEGF in hypoxia-conditioned media, which would be expected to support cell survival and proliferation, the increased levels of TSP-1 were able to override these protective effects. In support of this idea, TSP-1 has recently been shown to modulate the responses of endothelial cells to VEGF via interaction with the VEGF type 2 receptor .
TSP-1 has been studied previously in SSc and TSP-1 gene expression in skin correlates with severity of skin involvement in SSc and has been used in combination with other factors as a biomarker for the disease process . In SSc and control fibroblasts, TSP-1 is induced by culture under hypoxic conditions . In addition, TSP-1 activates the latent complex of transforming growth factor β (TGFβ) and maintains an autocrine loop of stimulation in SSc fibroblast by TGFβ . It seems likely that TSP-1-dependent effects occur in the hypoxic microenvironment of SSc and that TSP-1 contributes to the microvascular injury and failure of repair and/or angiogenesis, despite the elevation of VEGF in the disease.
Taken together, our data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues. Antiangiogenic factors can be targeted in future therapeutic approaches, both in SSc and in peripheral vascular disease, where TSP-1 is also induced and blocks microvascular repair .
bovine serum albumin
Dulbecco's modified Eagle's medium
enzyme-linked immunosorbent assay
foetal calf serum
human microvascular endothelial cell 1
quantitative real-time polymerase chain reaction
simian virus 40
vascular endothelial growth factor.
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