Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells

A major portion of the beneficial effect of mesenchymal stem cells (MSC) is due to the production of trophic and angiogenic factors by these cells, and one of the efforts to improve the therapeutic efficacy of these cells lies in enhancing this capacity. Since there is complement activation in all areas of tissue injury, and both C3a and C5a activate MSC, it was asked whether stimulation with C3a or C5a would upregulate the production of trophic factors by MSC. C3a caused significant up-regulation of various angiogenic factors, including VEGF, CXCL8/IL-8 and IL-6. In contrast there was no detectable production of the pro-inflammatory cytokines TNF-α and IL-1β in spite of nuclear translocation of NFκB. Although C5a also caused moderate up-regulation of angiogenic factors, the effect was borderline significant. Furthermore the production of angiogenic factors induced by C3a was of physiological relevance: Supernatants of MSCs cultured under serum-free conditions induced minimal tube formation of HUVECs as an in vitro measure of angiogenesis; tube formation was considerably enhanced, when supernatants from C3a-stimulated MSC were used, while C3a itself had no direct angiogenic effect on HUVECs. The signaling cascade responsible for the production of angiogenic factors by C3a or C5a could be defined as activation of the rho cascade which was necessary for nuclear translocation of NFκB p65 and of phospho-ERK1/2. Although rho was only transiently activated, inhibition of the rho or “downstream of it” of the NFκB pathway, prevented C3a-and C5a-induced up-regulation of angiogenic factors.

• MSC; C3a; C5a; Angiogenic Factor Produc-tion; Signaling Pathways

1. INTRODUCTION

Mesenchymal stem cells (MSC) are rare cells found in all tissues which are able to differentiate into all types of connective tissue lineages including osteoblasts, adipocytes and chondrocytes. In addition, these cells produce a variety of trophic and angiogenic factors which contribute to tissue regeneration [1,2] and possess immune suppressive properties [3,4]. Because of these properties MSC are starting to find clinical application in a variety of diseases ranging from myocardial infarction [5] to graft versus host disease [6]. However, the full regenerative potential of these cells has not been realized due to poor tissue homing and limited cell survival following transplantation. Currently various means of improving MSC homing [7], growth and angiogenic factor production [8,9] and in vivo survival [10,11] are being pursued as ways to improve the therapeutic efficacy of MSC. Chemotactic factors for MSC include several growth factors (bFGF, PDGF, IGF-1) [12,13], some chemokines [14-16], and the anaphylatoxins C3a and C5a [17].

C3a and C5a are small (Mr 8700-11,000) polypeptides released from their precursor proteins, C3 and C5, respectively by C3/5 convertases during complement activation. Both C3a and C5a are well known as chemotactic, oxidant inducing and degranulating agents for myeloid cells [18-23], but the response of leukocytes to C3a is considerably weaker and more transient than that to C5a [20,21,24]. In particular C3a does not cause leukocyte accumulation in vivo [24] which contrasts with the strong inflammatory response to C5a [18,25]. However in MSC the response to C3a was at least as strong as that to C5a [17] and apart from serving as chemoattractants both C3a and C5a protected from oxidative injury in MSC [17].

The C3a receptor (C3aR) and the C5a receptor (C5aR/ CD88) are G-protein coupled receptors (GPCR) which usually couple to Gi. Activation of the C3aR tends to result in transient cellular responses in most cell types [1926]. However, there have been a few exceptions, where C3a elicited a prolonged and strong response including mast cells [17,27] and as already mentioned MSC [17]. The C3aR contains two interesting features. First, it has an unusual, very long second extracellular loop, of which sequences adjacent to the transmembrane domains are important for C3a binding [28]. Second, an apparent nuclear localization signal sequence is located near the Cterminus of the C3aR, FRKKAR starting at amino acid 442 [29], but a functional significance for this observation has not been discerned. It has been noted, however that the C3aR in MSC, but not in other cell types tested can be translocated to the nucleus following the addition of C3a [17]. While GPCR activation at the plasma membrane induces mostly short term signaling events, nuclear translocation results in long-term effects including prolonged nuclear ERK1/2 activation [30] leading to transcriptional activation [31] and cell proliferation [32].

In monocytic cells, C3a and C5a activate rho in a Gidependent fashion, which leads to activation of NFκB downstream [33,34]. This in turn is responsible for transcriptional activation leading to the production of various inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-8/CXCL8. It is shown here that this signaling cascade is the same in MSC, but with a different outcome, since these cells appear to have developed mechanisms that suppress expression and/or processing of TNF-α [35] and IL-1β [36], while at the same time supporting increased expression of several angiogenic factors including VEGF, IL-6 and CXCL-8, thus converting a normally inflammatory signaling pathway into one that may contribute to tissue healing.

2. METHODS

2.1. Materials and Cell Culture

Human bone marrow MSC were supplied by the Tulane Center for Gene Therapy and cultured up to passage 5 in alpha-MEM (Life Technologies, Carlsbad, CA) containing 16.5% FCS (Atlanta Biologicals, Lawrenceville, GE). HUVECs were purchased from Lonza (Allendale, NJ) and grown in complete EGM (Lonza).

Natural C3a and recombinant C5a were purified as described [17] and were used at concentrations that had been shown previously to induce a maximal response [17].

The inhibitors PD98059, U0126, LY294002 and BayII- 7082 were purchased from EMD Biosciences (Gibbstown, NJ), Y27632 was obtained from Biomol (Enzo Life Sciences International, Plymouth Meeting, PA), Cell Permeable C3 Transferase protein inhibitor (CT04-A, Rho inhibitor) was supplied by Cytoskeleton (Denver, CO), and pertussis toxin by List Biological Laboratories

(Campbell, CA). Inhibitor concentrations were as follows: 50 μM PD98059, 10 μM U0126, 10 μM LY294002, 10 μM BayII-7082, 10 μM Y27632, 1 μg/ml C3 Transferase inhibitor, and 100 ng/ml pertussis toxin. Inhibitor pre-incubation times were 30 min except for the C3 Transferase inhibitor and pertussis toxin, which were added 20 hrs before cell stimulation. ELISA kits were from Biolegend (CXCL-8, IL-6, TNF-α, and IL-1β, San Diego, CA) or Peprotech (VEGF, Rocky Hill, NJ). Antibodies against total ERK1, NFκB p65 and Lamin A were from Santa Cruz Biotechnology (Santa Cruz, CA), all other antibodies (phospho-ERK1/2, phospho-Elk and total Elk) were from Cell Signaling Technology (Danvers, MA). The antibody arrays (RayBio Human Angiogenesis Antibody Array 1) were obtained from RayBiotech (Norcross, GA).

2.2. Subcellular Fractionation

To isolate nuclear fractions, cells were vortexed in hypotonic buffer (10 mM HEPES, pH 8.0, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 1 mM DTT, 0.2 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin) containing 0.5% NP-40, then microfuged at highest speed for 1 min. Supernatants representing the cytoplasmic fraction were retained, the pellets were washed 2 × with the same buffer, then resuspended in buffer B (20 mM HEPES, pH 8.0, 250 mM NaCl, 2 mM EDTA, 2 mM EGTA, 2 mM Na₃VO₄, 1 mM DTT, 0.2 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin), incubated on ice for 15 min followed by another 2 min centrifugation at 4˚C. The supernatants (nuclear fraction) were transferred to clean tubes containing 4 × gel sample buffer, boiled and separated on SDS-gels. The purity of the fractions was confirmed by Western blotting using lamin A as a nuclear marker and β-tubulin as a cytoplasmic marker.

2.3. Western Blots

The cellular fractions were resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 4% dry milk in TBS-Tween, and exposed to specific primary antibodies as described for each experiment. Antibody binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse secondary antibodies and enhanced chemi-luminescence (Super Signal West Dura, Thermo). Blots were re-probed with a second antibody, e.g. anti-ERK1 antibody to assure equal loading. ImageJ software was used to quantify results.

2.4. Rho Activation Assay

MSC at approximately 50% confluence were treated with 300 nM C3a or 50 nM C5a for the times indicated. Rho activity in total cell lysates was measured using a calorimetric ELISA-based assay (G-LISA, Cytoskeleton) according to the manufacturer’s instructions.

2.5. Fluorescence Microscopy

Cells were cultured on fibronectin-coated glass coverslips, stimulated for the indicated times with 300 nM C3a, 50 nM C5a or 50 ng/ml TNF-α, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 in PBS. After blocking with 2% FCS for 30 min at room temperature, cells were incubated overnight with antiC3aR (BD Biosciences), anti-phospho-ERK1/2 antibodies (Cell Signaling) or anti-NFκB p65 (Santa Cruz Biotechnology) at 4˚C, followed by staining with Alexa Fluor 488 anti-rabbit IgG or Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit IgG antibody (Life Technologies) in 2% FCS. Cells were then stained with DAPI (Sigma Chemical Co., St. Louis, MI) for 10 min and washed three times with PBS before mounting with AntiFade (Life Technologies). Images were taken on an Olympus FV1000 confocal microscope.

2.6. RT-PCR

MSC at about 60% confluence were cultured for 3 hrs in the presence or absence of 300 nM C3a or 50 nM C5a under serum-free conditions. RNA from MSCs was isolated with the RNeasy kit (Qiagen, Valencia, CA). Complementary DNA was synthesized from MSC RNA using Omniscript reverse transcriptase (Qiagen). The primers are listed under supplementary information. Amplification was performed for 35 cycles in a Perkin Elmer Cetus DNA thermal cycler.

2.7. ELISAs

MSC at about 60% confluence were cultured for 20 hrs in the presence or absence of inhibitors with or without 300 nM C3a or 50 nM C5a under serum-free conditions in α-MEM. Following the 20 hr incubation, cytokine concentrations were detected in the culture supernatants by commercial ELISA kits. Alternatively, the supernatants were used with an angiogenesis protein array (Ray Biotech, Norcross GA, angiogenesis array 1), which was developed according to the supplier’s manual.

2.8. In Vitro Angiogenesis Assay

HUVECs (passage 3-5) were used with the Millicell μ- Angiogenesis Activation Assay Kit (Millipore, Billerica, MA) following the kit’s instructions. Specifically, HUVECs were harvested with Accutase (Invitrogen) and resuspended in α-MEM containing 1% FCS and the same volume of test sample (serum-free α-MEM, serum-free α-MEM containing 300 nM C3a, serum-free α-MEM containing 50 nM C5a, serum-free α-MEM containing 100 ng/ml phorbol myristate acetate (PMA) as a positive control, the 20 hr supernatant of unstimulated MSC, the 20 hr supernatant of MSC stimulated with 300 nM C3a or the 20 hr supernatant of MSC stimulated with 50 nM C5a). The cells (10,000 cells/well) were layered over 10 μl of fibrin gel and incubated for 6 hr in a 37˚C tissue culture incubator. At this point the supernatant was carefully removed, the cells were overlaid with 10 ul of fibrin gel, the corresponding serum-free test sample was added again and the cells were incubated for another 24 hrs at 37˚C. Representative images were taken on a Nikon Eclipse TE200 microscope (Nikon Instruments, Melville, NY) with a Spot camera system (Diagnostics Instruments, Sterling Heights, MI).

2.9. Transwell Chemotaxis Assay

A single cell suspension of MSCs was loaded into the upper wells of 0.15% gelatin-coated Transwells (Costar, 8 μm pore-size, 2 × 10⁴ cells/insert) and pre-incubated with inhibitors for 30 min prior to the addition of stimulus. The lower wells contained 0.2% BSA in α-MEM and either no stimulus or doses of C3a (100 nM ) or C5a (15 nM), which had been found previously to induce maximal chemotaxis [17]. The assembled wells were incubated for 8 hrs in a tissue culture incubator, cells in the upper compartment were carefully removed, the filters were stained with DAPI and the transmigrated cells were counted on a Nikon Eclipse TE200 inverted fluorescence microscope (Nikon Instruments, Melville, NY) with a Spot camera system (Diagnostics Instruments). Background chemotaxis of unstimulated cells was defined as 100%.

3. RESULTS

3.1. Production of Angiogenic Factors by MSC Stimulated with C3a or C5a

Since the production of trophic and angiogenic factors is a major mechanism by which MSC support tissue repair, it was determined whether stimulation with C3a or C5a would up-regulate production of such factors by MSC. Semi-quantitative RT-PCR indicated that stimulation with C3a induced up-regulation of expression of genes encoding several angiogenic and growth factors including VEGF, IL-8/CXCL8, bFGF and TGF-β1 (Figure 1A). Up-regulation of several angiogenic and growth factors could further be verified on the protein expression level: This was first performed semi-quantitatively using a protein array that detects 20 angiogenic factors. C3a caused consistently increased expression of angiogenin, bFGF, gro-α/CXCL1, IL-6, CXCL8, MCP-1/CCL2, PDGF-BB, and VEGF (Figure 1B). Minor up-regulation was also seen for ENA78/CXCL5, leptin, the active form of TGF- β1, and VEGF-D (Figure 1B). No consistent effect was

seen for IFN-γ, IGF-I, PIGF, RANTES/CCL5, TIMP1 and TIMP2.

These semi-quantitative results were quantified by ELISA for CXCL8, IL-6 and VEGF as shown in Figure 1C. While C5a, which was used in parallel, also caused up-regulation of CXCL8, IL-6 and VEGF (Figure 1C), the increase was only statistically significant for IL-6.

Interestingly, this increase in angiogenic factor production was not accompanied by a concomitant increase in secreted TNF-α or IL-1β under any of the conditions used. TNF-α or IL-1β concentrations in MSC supernatants never reached the detection limit of the assay (5 pg/ml for TNF-α and 2 pg/ml for IL-1β).

3.1.1. Effect of C3aand C5a-Conditioned MSC Media on in Vitro Angiogenesis

In order to show the physiological relevance of the increased production of angiogenic factors by MSC stimulated with C3a or C5a, the same MSC supernatants were used in an in vitro angiogenesis assay, in which tube formation by HUVECs was evaluated. In the presence of α-MEM media, which was not conditioned by culture with MSC, the endothelial cells appeared as individual, separated cells (Figure 2A). The addition of PMA, which was used as a positive control, induced tube formation as expected (Figure 2B). Conditioned media derived from unstimulated MSC induced moderate alignment of cells into clusters (Figure 2C). Supernatants from MSC stimulated with C3a showed more pronounced capillary tube formation (Figure 2D) that was at least as prominent as seen with PMA (Figure 2B). The effect of the C3a-conditioned supernatant was not due to any direct effect of C3a on HUVECs, as C3a added to unconditioned α- MEM media showed no such effect (Figure 2E). The supernatant of C5a-stimulated MSC also caused moderate tube formation (Figure 2F). Again, direct addition of C5a had no such effect (results not shown).

3.1.2. Effect of Kinase Pathway Inhibitors on Production of Angiogenic Factors by MSC

In order to determine which signaling pathways were involved in C3aand C5a-mediated up-regulation of angiogenic factors, various pharmacological inhibitors which are known to block signaling pathways of C3a or C5a in other cell types were added prior to the addition of C3a or C5a to produce MSC conditioned media. Interestingly, a rho kinase inhibitor (Y27632) completely inhibited upregulation of VEGF and CXCL8 in MSC stimulated with either C3a (Figures 3A and B) or C5a (Supplementary

Table 1). The NFκB inhibitor BayII-7982 also largely inhibited increased production of VEGF and CXCL-8 (Figures 3A and B, and Supplementary Table 1), while inhibition of the ERK pathway with PD98059 (Figures 3A and B, and Supplementary Table 1) showed only a minor inhibitory effect.

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