1. Introduction
Extensive experimental and clinical evidence supports the link between complement system activation and the pathogenesis of diabetic vascular complications, including diabetic retinopathy (DR) and atherosclerosis [
1,
2]. The complement system is an effector for adaptive and innate immunity that is activated via three enzymatic cascades known as the classical, the mannose-binding lectin (MBL), and alternative pathways [
3,
4]. All three pathways eventually converge at the level of complement component 3 (C3) and thereafter share a common sequence of C5 convertase formation and generation of the membrane attack complex (MAC) (C5b-9). The formation of MAC results from the binding of C5b to complement proteins C6, C7, C8, and multiple molecules of C9. Once formed, MAC complex leads to osmotic imbalance and ultimately lysis of pathogens or cells. To prevent unintended damage to the host tissue by activated complement cascade, several complement regulatory proteins (CD55, CD46, and CD59) are anchored on the plasma membrane via a glyphosphatidylinositol. These regulatory proteins protect host cells from complement-induced self-cell damage [
3]. However, aberrant complement activation and impairment of complement regulatory proteins in pathological conditions can lead to MAC formation on host cells [
3,
4]. An increased level of MAC deposition found in the eyes of patients with DR when compared to eyes from non-diabetic subjects [
5], is suggested to be the result of both the reduced levels of complement regulatory proteins and continued activation of the alternative pathway [
6]. Moreover, glycosylation-induced impairment of functional activity of complement regulatory protein CD59 may also contribute to complement activation in pathologies such as diabetes [
7]. Increased MAC deposition in diabetic tissues has been implicated in the release of growth factors that promote abnormal cell proliferation in the vascular wall of vessels, thus contributing to the development of vascular proliferative disease [
8]. However, whether complement activation and MAC deposition participate in retinal endothelium damage remains unclear.
Intriguingly, complement components have been found to associate with extracellular vesicles, such as exosomes, in circulation [
9,
10]. Extracellular vesicles are small vesicles measuring 40–200 nm in diameter [
11], and are found in most biological fluids including blood, urine, cerebrospinal fluid, and ascites [
12]. Importantly, extracellular vesicles released from parental cells carry biological information such as nucleotides, proteins, and lipids that exert various effects on target cells. In fact, it has been suggested that extracellular vesicles may serve as a novel cell-cell communication method due to the heterogeneity of the cargo they carry from cell to cell [
13]. Recently, we have reported that plasma extracellular vesicles activate the classical complement pathway, and may contribute to MAC-induced retinal vascular permeability in diabetes [
14]. However, whether MAC induces cellular damage in the diabetic retinal vasculature remains unknown. In this study, we investigated the physiological mechanism in which diabetic plasma extracellular vesicles induce complement activation leading to MAC deposition and cellular impairment in human retinal endothelial cells (HRECs).
3. Discussion
Under normal physiological conditions, low-level complement activation and non-cytolytic MAC deposition are thought to be beneficial, serving as a mechanism to remove opsonized cellular debris and pathogens [
17] Complement activation and choriocapillaris loss in early AMD: Implications for pathophysiology and therapy, as well as maintaining the eyes immune privilege. However, under disease conditions such as those present in DR, continued complement activation and reduced level of complement regulatory proteins are suggested to be associated with increased MAC formation in the retinal endothelium lumen [
6]. Moreover, irreversible MAC deposition on endothelial cells leads to the release of mitogenic factors [
18], further supporting the rationale that complement activation is involved in the advancement of DR. Furthermore, circulating plasma extracellular vesicles have been shown to play a role in complement pathway activation leading to retinal vascular damage in vivo [
13]. No studies, however, have investigated the role that diabetic extracellular vesicles play in causing cytolytic damage to the endothelium in vitro. In this study, we demonstrate that diabetic extracellular vesicles induce complement activation and MAC deposition, which contributes to retinal endothelial cell damage. The results of this study suggest that extracellular vesicle-induced complement activation followed by MAC deposition could provide a novel mechanism for increased retinal vascular permeability and DR pathogenesis.
Previously, we have demonstrated that immunoglobulins are associated with extracellular vesicles in circulation and activate classical complement protein C1 in both a human and mouse model [
13]. In the current study, we demonstrated similar findings using rat plasma extracellular vesicles isolated by the ultracentrifugation method. These data suggest that activation of classical complement by IgG-laden extracellular vesicles occurs across species and is not dependent on the isolation method. Consistent with our previous finding, C1 activation occurs in a specific extracellular vesicle fraction (
Figure 2d), which further supports the rationale that a sub-population of extracellular vesicles may favor complement activation. Ultracentrifugation method isolated rat plasma extracellular vesicle samples had a reduced level of lipoproteins when compared to the plasma (
Figure 1a). This once again demonstrated that ultracentrifugation is the method of choice for a higher purity of extracellular vesicle isolation [
19] and it, therefore, is ideal for extracellular vesicle characterization. However, the ultracentrifugation method is not quantitative due to the inherent variability of this multi-step process and is thus not suitable for comparing samples between control and diabetic groups. The ExoQuick isolation method was consequently the alternative method of choice to compare the number of vesicles between control and diabetic extracellular vesicles.
Ogata N. et al. demonstrated that an increase of extracellular vesicles in diabetic plasma could contribute to the acceleration of DR progression [
20,
21]. We have recently reported, using a novel extracellular vesicle quantification method in mice, that diabetes causes an increase in the number of circulating extracellular vesicles found in venous blood [
13]. In agreement with this finding, our present study demonstrates that the number of extracellular vesicles is also increased in the arterial blood of STZ-induced diabetic rats when compared to controls (
Figure 3a). However, in contrast with our previously reported venous samples, the diameter of the extracellular vesicles increased more in diabetic arterial circulation than in control. Additionally, arterial extracellular vesicles contain a wider range of CD63 positive vesicles in the Opti-prep density gradient (
Figure 1b) and a higher value of polydispersity (data not shown) than our previously reported venous extracellular vesicles. Due to these findings, we speculate that extracellular vesicle populations may change as they pass from arterial to venous vascular beds and shift the density of the extracellular vesicles. Interestingly, extracellular vesicle markers TSG101 and CD63 showed no difference between control and diabetes in arterial blood (
Figure 3c) while the total number of extracellular vesicles increased in diabetes. This suggests that TSG101 and CD63 positive extracellular vesicles may not contribute to the increased number of vesicles in diabetic atrial blood. It is also possible that ExoQuick isolated plasma extracellular vesicles contain other vesicles such as lipoproteins [
22], which could contribute to the vesicle differences seen between the control and diabetic condition. Due to the rat plasma containing these varied populations, OptiPrep gradient fractionation is needed after the ExoQuick method to isolate the IgG-laden extracellular vesicles and observe their effect in future studies. Our current study demonstrated that the IgG level was higher in isolated diabetic arterial extracellular vesicles than in control extracellular vesicles. In agreement with the previous study [
13], C1 activation at the vesicle fractions had a higher density (1.24 g/mL and above), where ALIX and CD9 expression were high, but low on CD63 expression. Heterogeneity of the extracellular vesicles on marker expression and the functional level was reported by several groups and is an active area of investigation [
11,
12,
13]. Importantly, in this study, we demonstrated for the first time that diabetic extracellular vesicles have higher C1 activity than control extracellular vesicles.
In physiological states, a low level of complement activation in the eye is tightly regulated and MAC deposition on the surface of cells is normally rapidly removed. Endothelial cells are targeted continuously by activated complement cascade, and MAC deposition is rapidly eliminated via endocytosis, protecting the cells from cytolytic destruction [
23]. A similar mechanism was reported to also occur in retinal pigment epithelial cells (RPEs) [
24]. Moreover, it has been suggested that, in vitro, MAC-induced mitogenesis contributes to focal tissue repair or pathological cell proliferation [
25]. In the present study, we observed that MAC deposition occurred in the retina of STZ-induced diabetic rats (
Figure 6); this is consistent with previous studies in the eyes of diabetic patients and animal models [
6,
7]. Besides, we show evidence suggesting that diabetic rat plasma induces robust MAC deposition and cytolytic damage in HRECs, but not with control plasma (
Figure 4). We reason that in the early stages of the DR, increased non-cytolytic retinal vascular MAC deposition contributes to signal focal tissue repair. As the disease advances, sustained complement activation accompanied by complement regulatory proteins impairment [
6] and deficient of retinal tissue repair processes [
26] heighten the MAC deposition, and this may contribute to cytolytic damage and increased retinal permeability. Previously, Kim D et al. demonstrated that complement regulatory proteins are homologous restricted and are less effective on complement targets from different species [
27]. Moreover, gal-(alpha 1-3)-gal epitopes on endothelial cells have a high affinity to natural antibodies, such as IgG from different species and favor classical complement-induced xeno-organ rejection [
28]. These reports suggest that in our study, surface-expressed complement regulatory proteins on HRECs might be less effective in protecting cells from rat extracellular vesicle-induced complement activation. Furthermore, HRECs might be sensitized by rat extracellular vesicle-associated and/or extracellular vesicle-unassociated immunoglobulins, which favor classical complement activation. Additionally, in diabetic plasma, hyperglycemia is usually accompanied by an elevated level of inflammatory cytokines and chemokines; this pro-inflammatory environment, coupled with the extracellular vesicles′ ability to carry proteins, nucleotides, and lipids makes extracellular vesicles more likely to facilitate cellular damage. Thus, we speculate that in diabetic rat plasma there are factors other than extracellular vesicle-induced complement deposition that may contribute to HREC damage. Interestingly, when we deplete extracellular vesicles from the diabetic plasma, complement-induced MAC deposition and cytolysis of HRECs were prevented. This suggests that plasma extracellular vesicles, in part, contribute to complement-dependent retinal cellular injury. However, the addition of diabetic plasma extracellular vesicles back into the diabetic extracellular vesicle-free plasma environment did not result in a full recovery of the cytotoxic effect, suggesting that extracellular vesicle isolation methods employed in our study may have inhibited proteins that are important for cellular cytotoxicity. These results highlight the importance of continued investigation and development of extracellular vesicle isolation techniques.
Recently, complement activation by cholesterol crystals was shown to contribute to vascular damage in human atherosclerosis and animal models [
16,
29]. To rule out the role of cholesterol crystals in HREC activation and MAC deposition, we performed SEM under non-dehydrating conditions to preserve potential cholesterol crystals. We observed the extracellular vesicles of expected 100–200 nm diameter in both control and diabetic extracellular vesicle preparations, and lipid particles ranging from 5~700 nm in blood plasma. There were no cholesterol crystals in control or diabetic plasma, or extracellular vesicle preparations used for HREC treatments.
In summary, our study demonstrates that diabetic rat plasma extracellular vesicles activate a greater level of classical complement protein C1 than control plasma extracellular vesicles. Activation of the complement cascade, in turn, contributes to MAC deposition and cytolytic damage of the retinal endothelial cells in diabetes. These findings may provide a novel mechanism that could contribute to endothelial cell dysfunction and the advancement of DR pathogenesis. Future research directions may also be highlighted.
4. Materials and Methods
4.1. Animal Studies
All animal procedures were in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved and monitored by IACUC at Michigan State University (AUF Busik08/17-151-00, approval date: 28/08/2017–28/08/2020). Male Sprague-Dawley rats (237–283 g) were made diabetics with a single intraperitoneal injection of streptozotocin (STZ) (65 mg/kg) (Sigma Aldrich) dissolved in 100 mM citric acid (pH = 4.5) [
30]. Body weight and blood glucose concentration were monitored biweekly and blood glucose concentration was maintained in the 20 mmol/L range. Rats with 7 weeks since onset of diabetes were used in this study.
4.2. Cell Culture
Primary human retinal endothelial cells (HRECs) were prepared from postmortem tissue obtained from National Disease Research Interchange, Philadelphia, PA, and EverSight Midwest Eye-Banks, Ann Arbor, MI. Primary HREC were isolated and cultured as previously described [
31]. Passages 3–6 were used in the experiments at 80–90% confluence in 10% FBS media before treatments.
4.3. Western Blot
Western blot analysis was performed as previously described, with the following antibodies at dilution 1:1000: anti-CD63, anti-CD9 and anti-TSG101 (SBI, Cat.NO.EXOAB-CD63A-1, EXOAB-CD9A-1 and EXOAB-TSG101-1), anti-ALIX (AbCam, Cat. No. ab117600), anti-C1q (CompTech, Cat. No. A200), anti-C1s (Quidel, Cat. No. A302). IRDye Donkey anti-rabbit or anti-goat was used as secondary antibodies (Rockland, Cat. No. 611-731-127) (LI-COR, Cat. No. 925-32213). Immuno-reactive bands were visualized by using the Odyssey digital imaging program.
4.4. Blood Sample Collection
Blood was collected from inferior vena cava or from tail artery of the animal into tubes with EDTA (SARSTEDT microvette-300). Plasma was harvested via centrifugation and either used immediately or aliquoted into 1.5 mL micro-tubes and stored in −80 °C.
4.5. Extracellular Vesicle Isolation
Sequential ultracentrifugation method for extracellular vesicle extraction was conducted as previously reported [
32]. In short, 0.5 mL of rat plasma was mixed with an equal volume of PBS, and then a series of low speed centrifugations of the supernatant were conducted to remove cellular debris. The supernatant was then filtered via a 0.22 µm filter, and extracellular vesicles were precipitated by spinning at 100,000 g for 2 h at 4 °C in a SORVALL M120SE Micro-Ultracentrifuge (S55S-1155 Rotor, SORVALL). The extracellular vesicle pellet was washed by re-suspension with PBS and spun down at 100,000 g before further analysis. ExoQuick (SBI, EXOQ5A) isolation was conducted based on manufacturer’s protocol.
4.6. Extracellular Quantification
Extracellular vesicle quantification was conducted based on previously reported procedures by combining dynamic light scattering (DLS) and static light scattering (SLS) technologies via Zetasizer Nano NZ (Malvern Instruments Ltd., Malvern, UK) [
13].
4.7. OptiPrep Density Gradient Extracellular Vesicle Purification
Discontinuous iodixanol gradient was used to further purify the extracellular vesicle solution. Purification of extracellular vesicles by OptiPrep density gradient was done as previously described [
15].
4.8. C1q Binding Assay
C1q binding assay was performed based on previously published procedures with modifications [
33,
34]. Isolated rat plasma extracellular vesicles (from 0.5 mL plasma) were re-suspended in 100 uL HEPES buffer (150 mM NaCl, 2 mM CaCl
2, 20 mM HEPES, pH 7.0) and incubated with 2 ug of C1q (CompTech. A099) for 30 min at 37 °C. After the incubation, extracellular vesicles were isolated via ultracentrifugation, purified by OptiPrep density gradient, and then analyzed by Western blot.
4.9. C1 Activation Assay
The ability of extracellular vesicles to induce C1 activation was measured using an in vitro assay as previously published with modifications [
15]. Extracellular vesicles were isolated from 0.5 mL of rat plasma, re-suspended in C1 activation assay buffer (50 nM triethanolamine-HCL, 145 mM NaCl, 1 mM CaCl
2, pH 7.4) and incubated with C1 complex (0.25 uM) (CompTech, A098) in the presence or absence of C1 inhibitor (INHC1) (CompTech, A140) for 90 min at 37 °C. The reaction mixtures were incubated on ice for 10 min, submitted to OptiPrep density gradient purification to isolate extracellular vesicles, and the activation of C1s was analyzed by Western blot.
4.10. LDH Assay
Cytotoxicity of the cells was quantified using an LDH assay kit, following the manufacturer’s protocol (Abcam, ab102526). First, 105 HRECs/100 µL were plated onto a 0.1% gelatin coated 96-well plate, incubated at 37 °C for 48 h, and then treated with 20% of control or diabetic rat plasma at 37 °C for 6 h. Additional conditions were created to determine the contribution of extracellular vesicles vs. other plasma components to cell cytotoxicity. Vesicle-removed control or diabetic plasma, and vesicle-removed control or diabetic plasma with control or diabetic extracellular vesicles added back in were used. After the treatment with plasma, the media was collected into a 1.5 mL Eppendorf tube and spun down at 10,000 g for 15 min at 4 °C. Supernatants (50 µL) were transferred into a 96 well plate and a mixed detection kit reagent (50 µL) was added to each well along with the NADH standard. The absorbance (450 nm) was taken on a micro-plate reader in a kinetic mode every 3 min for 30 min at 37 °C. LDH activity of the samples was calculated: LDH activity (milli-units/mL) = amount of NADH (nmol)/(Reaction Time × Sample Volume) × Dilution Factor. LDH concentration from no cell conditions (
Figure S1) were subtracted from each condition before calculation of cytotoxicity.
4.11. Immunocytochemistry
Immunocytochemistry was performed as previously described using anti-MAC (C5b-9) antibody (Abcam, ab55811) at 1:100 in PBS with 1.5% BSA overnight at 4 °C, followed by chicken anti-rabbit Alexa Fluor 594 secondary antibody (1:500) and DAPI (Sigma-Aldrich, St.Louis, MO, USA) nuclei counterstaining. The slides were analyzed using Nikon TE2000 microscope equipped with Photometric Cool-SNAP HQ2 camera. All images were taken with matched exposure time for experimental and control slides by using the MetaMorph imaging software (Molecular Devices, Downington, PA, USA).
4.12. Electron Microscopy
The blood plasma and extracellular vesicles were prepared for scanning electron microscopy (SEM) as previously described, omitting standard ethanol dehydration and critical point drying steps [
16]. The samples were smeared on cover slips, mounted on SEM stubs, fixed with osmium tetroxide vapor and gold coated in an EMSCOPE SC500 sputter coater (Emscope, Ashford, UK) followed by examination using a JEOL SEM (model JEOL-6610LV, JEOL Ltd., Tokyo, Japan).
4.13. Statistics
A Student paired t-test was used to analyze data with two groups. In experiments with multiple group comparisons, one-way ANOVA with post-hoc analysis by Tukey’s range test (GraphPad Prim 7, GraphPad Software, San Diego, CA, USA) was used. All values are expressed as mean ± Standard Deviation. p-values below 0.05 were considered significant.