Complement Activation During Ischemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling

Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRβ+/NG2+ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFβ pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.

inTrODUcTiOn Ischemia/reperfusion (I/R) injury remains an unavoidable consequence after renal transplantation and the principal cause of delay graft function (DGF) (1). After brain death, the decreased blood flow induces a persistent rarefaction in peritubular capillaries (2,3), whereas the following reperfusion exacerbates the pro-inflammatory response by activation of complement and coagulation (4). Pharmacologic treatments to prevent graft deterioration after I/R are currently lacking. Recently, genetic fate-mapping studies have identified pericytes as the major source of scar-forming myofibroblasts during progressive chronic kidney disease (5)(6)(7)(8). Pericytes are mesenchymal-derived cells embedded in the capillary basement membrane and in direct contact with endothelial cells (9). Pericytes contribute to microvessel stability and show regeneration potential; renal pericytes regulate cortical and medullary flow by contracting or dilating in response to various stimuli released by the neighboring endothelial and tubular cells (10). Interestingly, data on cerebral ischemia showed that pericytes died "in rigor," causing an irreversible constriction of capillaries that exacerbates the hypoxia (11,12). Renal pericytes are PDGFRβ + /NG2 + cells that during their abnormal transdifferentiation in myofibroblasts upregulated the pro-fibrotic marker αSMA. The physiological role of the receptor tyrosin kinase PDGFRβ is to bind the platelet-derived growth factor B (PDGF-B) released by endothelial cells. The PDGFRβ signaling promoted the pericytes activation, migration, and the recruitment to the vascular wall of newly formed blood vessels. Nerve/glial antigen 2 (NG2) is a proteoglycan associated with pericytes during vascular morphogenesis (13).
Complement plays a pivotal role in renal I/R injury mediating tissue damage and amplifying innate and adaptive immune response (14)(15)(16)(17). C1-esterase Inhibitor (C1-INH) blocks complement activation of the classical, lectin (14,18,19), and alternative pathways (20)(21)(22). Currently, C1-INH is used as treatment for hereditary angioedema (23), but several studies are evaluating the therapeutic potential in renal transplantation (24)(25)(26). In previous studies, we demonstrated that C1-INH is able to prevent the C5b-9 deposition along peritubular capillaries, limiting endothelial dysfunction and renal fibrosis during I/R (18,27). The aim of our study was to investigate the involvement of complement in pericyte activation in the early phase of I/R injury.

MaTerials anD MeThODs animal Models
Animal studies were carried out under protocol approved by Ethical Committee of the Italian Ministry of Health. Briefly, I/R was induced in pig by clamping the renal artery for 30 min followed by reperfusion, as described previously (18). A biopsy was performed before ischemia (T0). Pigs were divided into two groups: control (CTRL, n = 5, vehicle infused) and C1 Inhibitor treated group (C1-INH, n = 5). Five minutes before the beginning of the reperfusion, rhC1-INH was injected in the ear vein (500 U/kg). Biopsies were performed at 15, 30, and 60 min and 24 h after reperfusion. All animals were sacrificed 24 h after the procedure. Controlateral kidney was not removed for ethical concerns. A mouse model of renal bilateral I/R was performed in C5aR1 −/− mouse with C57BL/6 backgrounds, as previously described (28).

analysis of Peritubular capillaries area
The peritubular capillaries area was calculated by Image J software. The cortical area of the entire biopsy acquired by Aperio ScanScope was analyzed in a stepwise fashion as a series of 10 consecutive fields, avoiding the arterioles, venules, and capillaries, which has a diameter upper than 50 µm. Values from all consecutive images for each biopsy were averaged.

MTT assay
Cultured pericytes proliferation was measured by MTT Cell Proliferation Assay Kit, according the manufacturer instructions (Sigma-Aldrich). Briefly, 2 × 10 4 cells/well were seeded in a 96-well plate, and then cells were treated with C5a, TGFβ1 (as indicated), PDGFBB (10 ng/ml) for 24 h.
rna extraction and qPcr analysis RNA from pericytes was extracted using the miRNeasy Kit (Qiagen), 500 ng of total RNA was retrotranscribed with QuantiTect Kit (Qiagen). qPCR was carried out with SsoAdvanced™ Universal SYBR ® Green Supermix (Biorad) and the Light Cycler@96 (Roche). Primer list sequence in Table 1.

statistical analysis
Graphs were displayed using GraphPad Prism Software 5. Data were expressed as median ± interquartile range (IQR) and compared with a Mann-Whitney test for tissue immunostainings. For FACS, qPCR, MTT, and WB data were expressed as the mean ± SD. Statistical analysis was assessed using unpaired Student's t-test. A p-value of <0.05 was considered significant.

c1-inh Treatment Preserved Pericytes Phenotype after renal i/r injury
To investigate the possible dysfunction of renal pericytes during I/R injury, biopsies were analyzed for the expression of PDGFRβ, a marker of pericyte (Figure 1). Under normal condition (T0, Figure 1A), PDGFRβ + cells were detected in interstitial peritubular capillaries ( Figure 1A, zoom1), in arterioles ( Figure 1A, zoom2), mesangial cells, and Bowman's capsule (Figure 1A, zoom3). I/R injury caused a significant decrease in PDGFRβ expression of pericytes in peritubular capillaries, a process that in the CTRL group began after 30 min and persisted until 24 h after reperfusion (Figures 1B,D,F). Treatment with C1-INH was unable to prevent early PDGFRβ downregulation (Figures 1C,E) but gave a significant preservation of peritubular PDGFRβ expression at 24 h after I/R injury ( Figure 1G compared to Figure 1F). Notably, the PDGFRβ expression of mesangial cells was not significantly down regulated. Next, we used PDGFRβ and NG2 co-expression to specifically label pericytes (Figure 2). In swine kidney, pericytes markers were localized in the interstitial peritubular capillaries (Figure 2A). We found that all the perivascular NG2 + cells were PDGFRβ + ; on the contrary, we found that mesangial cells were PDGFRβ + /NG2 − . After 24 h of I/R, the total number of PDGFRβ/NG2 double positive pericytes significantly decreased; in accordance with Figure 1F, PDGFRβ + /NG2 + cells were barely detectable in the interstitial peritubular capillaries after I/R (Figures 2B,E). In contrast, C1-INH treated pigs were protected in pericytes phenotype as shown by a significant recovery in the number of PDGFRβ + /NG2 + cells in the interstitial peritubular capillaries regions. Interestingly, after 24 h from the C1-INH treatment, other PDGFRβ + /NG2 − cells (i.e., vascular smooth muscle cells) were protected from the PDGFRβ downregulation.
To further confirm the complement deposition at peritubular capillary level, we investigated the co-localization of PDGFRβ with C3 and C5b-9 after 30 min of reperfusion (Figures S1 and S4 in Supplementary Material) on frozen renal tissue. We found C3 and C5b-9 deposition around peritubular regions after 30 min; interestingly, C1-INH significantly counteracted this

i/r injury Did not affect Pericytes Viability In Vivo
To study the possible occurrence of pericyte apoptosis during I/R injury as observed in cerebral ischemia (12), we stained 30 min, 60 min, and 24 h serial sections for PDGFRβ and for the active form of Caspase 3 (Casp3) (Figure 3). After 30 and 60 min from reperfusion, no PDGRFβ/Casp3 double positive cells were detected in peritubular capillaries ( Figures 3A,B arrowheads). As previously shown (18), apoptosis occurred predominantly in tubular epithelial cells ( Figures 3A,B right, brown nuclei) ( Figure  S1A in Supplementary Material), and not in pericytes that were PDGFRβ + /Casp3 − . In addition, we investigated whether renal pericytes proliferation could be detected, labeling serial sections for PDGFRβ and Ki-67, an antigen that marked nuclei in G1, S, and G2 cell cycle phases (29). Remarkably, 24 h after I/R injury, no Ki-67 positive cells could be found in interstitial peritubular capillaries (Figures 3C,D). In conclusion, in our model, cellular apoptosis and proliferation occurred at the level of tubular epithelial cells and not within cells of peritubular capillaries ( Figure  S1B in Supplementary Material).
complement Modulation abrogated i/r injury-induced PMT and attenuated capillary lumen reduction Next, we investigated the possible occurrence of PMT after I/R. Normal kidney showed αSMA expression predominately in smooth muscle cells (wall of renal arteries, Figures 4A,D, dotted arrow). Before ischemia, we found PDGFRβ + /αSMA − pericytes in interstitial peritubular capillaries (Figures 4A,B). After 24 h from I/R injury, perivascular cells upregulated αSMA together with an intense reduction in PDGFRβ expression, indicating PMT (Figures 4C,D). The co-localization of these two markers was more evident in arterioles and peritubular capillaries as shown in Figure 4D. Mesangial cells, which originate from the same FOXD1 + embryonic mesenchymal precursors of pericytes, expressed PDGFRβ (30,31). However, PDGFRβ expression by mesangial cells remained unaffected and no increase of PDGFRβ + /αSMA + was detected in glomerular cells ( Figure 4H). C1-INH treatment significantly reduced the number of PDGFRβ + /αSMA + cells in the peritubular capillaries, preserving the physiological pericytes phenotype (Figures 4E-G). These data demonstrate that that inhibition of C1 activity is associated with decreased PMT. Interestingly, we assessed the occurrence of PMT in a mouse model of renal I/R injury. In the Wtype sham, we  Figure 5A). One day after reperfusion, a significant reduction of PDGFRβ and an increase of aSMA in the peritubular capillaries were observed in the Wtype (Figure 5B). On the contrary, C5aR1 −/− mice showed significantly lower number of PDGFRβ + /αSMA + cells compared with the Wtype (Figures 5C,I).

c5a induced PMT Without affecting Pericytes Viability In Vitro
To validate our findings in vitro, we next evaluated the PDGFRβ expression in pericytes culture stimulated with the complement anaphylotoxin C5a and TGFβ, a classic mediator of PMT (7,32). After C5a stimulation, we found that the number of PDGFRβ + -pericytes significantly decreased indicating the This activation persisted even after 48 h from stimulation (data not shown). By using the AnnexinV/Propidium Iodide and MTT test, we also found that pericytes did not undergo to early and late apoptosis upon C5a activation (Figure 6C, early apoptosis: C5a 14.57 ± 0.82% versus Bas 11.19 ± 1.52%, ns, Figure S2A in Supplementary Material) nor downregulated their proliferative response (Figure 6D). On the contrary, TGFβ stimulation upregulated the percentage of early apoptotic cells during pericytes activation.
We also observed that C5a-stimulated pericytes acquired spindle-like shape morphology similar to fibroblasts. Performing immunofluorescence analysis, we found an increased expression of αSMA in stress fibers (PDGFRβ/αSMA, Figure 7A), indicating the acquirement of a contractile phenotype. Moreover, the morphologic changes were accompanied by increased collagen I protein synthesis (Figure 7B, C5a 24 h: 63.17 ± 8.22% versus basal: 19.86 ± 15.07%, p < 0.05) as well as connective tissue growth factor mRNA expression ( Figure 7C). Interestingly, C5a increased metalloproteinase MMP9 protein level and ADAMTS1 gene expression, which are usually involved in pericytes    Finally, we evaluated the modulation of Id2, a critical DNAbinding protein implicated in the regulation of the profibrotic/ mesenchymal terminal differentiation (34)(35)(36). As expected, Id2 was highly expressed in normal cultured pericytes. By 6 h from C5a stimulation, we found a significant downregulation of Id2 in pericytes undergoing PMT; the downregulation was still detectable at 18 h and was comparable to the effects of TGFβ, a negative modulator of Id2 ( Figure 7D).

c5a signaling induces PMT by canonical and non-canonical TgFβ Pathway via perK
To identify the intracellular signaling involved in the C5a-induced PMT, cultured pericytes were incubated with complement anaphylotoxin and TGF-β1 (Figures 8 and 9). TGFβ contributes to renal fibrosis by the activation of canonical (SMAD 2/3) pathway and non-canonical [mitogen-activated protein kinase (MAPK)] pathways (37) (Figure S3 in Supplementary Material). Since also C5a, by interaction with the C5aR, induces the activation of ERK/MAPK pathway (38-40), we evaluated pERK protein modulation, as a possible common mediator of TGFβ and C5a signaling. We found by FACS analysis that pericytes expressed the C5aR at cytoplasmic and membrane level ( Figure 8A). In addition, pericytes significantly upregulated the C5aR1 mRNA, which increased after 18 h of stimulation ( Figure 8B). As shown in Figure 8C, C5a increased ERK phosphorylation after 15 and 30 min.
Next, we investigated whether the C5aR-induced signaling could play a role in promoting the PMT. We used anti-C5aR specific neutralizing antibody to inhibit the C5a binding to C5aR. Anti-C5aR prevented the increase of collagen I induced  by C5a exposition (Figure 8D, bottom right). These results indicate that pericytes expressed the C5aR and that C5aR activation via pERK (non-canonical TGFβ pathway) might promote PMT. To further validate that pERK pathway was also pivotal in TGFβ-canonical signaling, we evaluated the effect of C5a stimulation on SMAD2/3 phosphorylation. First, we found the C5a increased the amount of pSMAD2/3 at 15 and 30 min (Figure 9A). Further time course at 6 and 18 h revealed that C5a led to a persistent increase of total SMAD2/3 complex ( Figure 9B). Finally, we tested the effect of SC1 (Pluripotin) on C5a-induced PMT. SC1 is a dual kinase (ERK1, MAPK3) inhibitor that blocks ERK1/2 phosphorylation of at Thr-202/ Tyr-204 (41). Analysis of ERK phosphorylation showed an inhibition at the concentration of 1 µM for 24 h (0.26 ± 0.18fold compared to untreated cells 2.4 ± 1.18); higher concentrations (3-5 µM) interfered with cellular viability and were not considered. Pretreatment of pericytes with 1 µM SC1 for 24 h reduced ERK phosphorylation (Figure 8C), blocked C5a-induced SMAD3 phosphorylation at 15 min (SC1 1 µM SC1 for 24 h + C5a 15 min: 0.69 ± 0.32 compared to C5a 15 min: 1.32 ± 0.25) ( Figure 8D) and significantly reduced the C5a-induced collagen I production at 12 h (SC1 1 µM SC1 for 24 h + C5a 12 h: 33.10 ± 12.15 compared to C5a 12 h: 8.34 ± 4.39) (Figure 8D, left bottom). These data support the role of C5a in promoting PMT by the activation of both TGFβcanonical and non-canonical pathway.

DiscUssiOn
In the present study, we demonstrated that inhibition of the complement system in I/R injury prevents the occurrence of PMT and the reduction of peritubular capillaries lumen areas. In particular, C5a had a pro-fibrotic activity driving pericytes toward a maladaptive dysfunctional phenotype by modulation of pERK activation.  Pericytes are mesenchymal-derived cells that interact with endothelial cells, releasing trophic factors such as VEGF and PDGF-BB (42). Recently, several studies demonstrated that pericytes are one of the myofibroblast precursors during development of tissue fibrosis (6,(43)(44)(45). Our results showed that complement is involved in transdifferentiation of pericytes in the early phases after kidney transplantation. Complement system is a key player in renal I/R injury (17,46,47), and C1-INH, a serine protease inhibitor used for the therapy of hereditary angioedema (23) might offer a new strategy for the prevention of I/R injury (25,48,49). Previously, it has been shown C1-INH treatment significantly prevents fibrosis, improves early and long-term renal function (26) and protected from TGFβ pathway activation (49). In our swine model of I/R, we found at early time after reperfusion (30 min) the deposition of complement components (i. e C4d, C3c, and C5b-9), along the peritubular capillaries (18), the areas where pericytes niche are localized. We also found the co-localization of PDGFRβ together with C3 ( Figure S1 in Supplementary Material) and C5b-9 ( Figure S4 in Supplementary Material). In accordance, starting from 30 min, the downregulation of PDGFRβ in our model began exactly along peritubular capillaries, without involvement of PDGFRβ expression in mesangial cells and larger arteries. The treatment with C1-INH, reduced the C4d peritubular deposition (18), and significantly restored pericytes markers expression, thereby accelerating the pericytes recovery. Even if pericytes identification requires transmission electron microscopy and fate-tracing analysis (5) several studies suggested that a PDGFRβ + perivascular cell population is involved in collagen release, since specific PDGFRβ blocking reduced fibrosis development (32). Additionally, NG2 (50-52) was used to specifically label pericytes. We demonstrated a significant reduction in PDGFRβ/NG2 double positive cells after 24 h of I/R. Interestingly, our data are in line with Hosaka and colleagues (53) showing that in the PMT occurring during the tumor growth and metastasis, the loss of PDGFRβ and NG2 is not due to pericytes death nor proliferation. We found that renal I/R injury did not induce apoptosis of pericytes but their activation characterized by a maladaptive response not leading to cellular death but transdifferentiation toward a myofibroblast phenotype. In accordance, complement system did not affect pericytes viability in vitro. Furthermore, our data on renal pericytes are different compared with a model of cerebral ischemia in vivo, were pericytes died by apoptosis in rigor mortis and induced an irreversible constriction of micro vessel (12). Another difference in our data regarded the pericyte proliferation in the early stage of I/R. Using a rat model of I/R (54) and a transgenic reporter mice to determine the contribution of pericytes to fibrosis, previous reports described the increased proliferation of pericytes, starting from 48 to 72 h after injury (5). This difference might be explained by the fact that our analysis was conducted at 24 h; in addition, we analyzed a swine model.
Several studies revealed the importance of kidney pericytes for peritubular capillary integrity (55) and the microvascular rarefaction following ischemic acute kidney injury (AKI) (56,57). After I/R, microvessels showed CD31 reduction and αSMA increase indicating the occurrence of endothelial-to-mesenchymal transition, which also contributes to kidney fibrosis (2,27,53); in this contest, αSMA, a marker of activated myofibroblasts, amplifies cell contractility with reorganization of stress fibers (9,58). Here, we demonstrated that αSMA increase is also associated to microvascular pericytes. In accordance with Gomez and colleagues (59), we hypothesized that PMT might lead to direct constriction of vessels with reduction of capillaries density and lumen area. This process has been described in cerebral I/R injury where pericytes led to irreversible constriction of capillaries, exacerbating tissue hypoxia (11,12). Interestingly, we found that treatment with C1-INH was capable to maintain the capillary lumen area by counteracting PMT. We recognized that C1-INH, beyond targeting classical and lectin pathway can inhibit the contact, coagulation, and fibrinolytic pathway involved in blood flow dysfunction (24). This can result in a reduced thrombi formation and a systemic improvement of vascular stability. However, PMT inhibition by C1-INH could provide a new mechanism to preserve graft from capillary rarefaction and reduction of lumen area.
Ischemia/reperfusion injury is primarily mediated by complement activation, with C5a playing a pivotal role also in inflammation response and allograft rejection (14,15,17,60,61). In this paper, we demonstrated for the first time that C5a can induce PMT with important pro-fibrotic effects. We stimulated cells with C5a because represents the most potent pro-inflammatory and chemotactic mediator (62) with specific receptors demonstrated at the level of renal resident cells. Nevertheless, despite the C5a pivotal pathogenic role, in clinical trials, the C5 inhibition by the human monoclonal antibody Eculizumab has been shown not to be sufficient to prevent DGF as well as antibody-mediated rejection (63,64). Specifically, even after Eculizumab treatment, a residual C5 activity has been demonstrated (65). This could explain why not all patients benefit of an anti-C5 therapy in C3-mediated kidney diseases or during strong complement activation (24). Therefore, strategies that act upstream of C5 activation to prevent opsonization and generation of C3 activated products (i.e., C3a, iC3b, C3b, and later C5a) have been evaluated (24). As endogenous serine protease inhibitor, C1-INH has an excellent safety compared to Eculizumab (66,67) and indirectly inhibits the release of the reactive late component C5a (68).
The C5a/C5aR pathway has extensively shown to cause recruitment of neutrophils and macrophages and exacerbate tubular injury in acute kidney injury. C5aR deficiency on renal cells or circulating leukocytes can significantly ameliorate renal injury (14,16,28,39,69).
The C5a-induced PMT was characterized by: the acquirement of αSMA stress fibers, the production of extracellular matrix proteins as collagen I, the downregulation of the antifibrotic BMP7-Id2 signaling (70)(71)(72), and finally by the activation of TGFβ canonical and non-canonical pathway (70)(71)(72)(73)(74)(75)(76)(77). Our data connecting complement activation with fibrosis are in accordance with other disease model such as lung fibrosis where complement might lead to SMAD2/3 dependent and independent pathway activation, shifting the initial acute inflammatory response in a chronic profibrotic state (77).
We also analyzed ERK activation at early times after C5a stimulation, since ERK is a common downstream mediator of C5aR, TGFβ non-canonical signaling, and a possible inducer of TGFβ canonical pathway (73,75) (Figure S3 in Supplementary Material). First, we showed that human pericytes expressed the C5aR, both at cytoplasmic level and on membrane surface. Second, by blocking the C5aR, we demonstrated that C5aR signaling is involved in collagen I release. As observed by other group (77), C5a can activate SMADs proteins, independently from TGFβ. In this signaling, ERK could act as bifurcation point to induce both the non-canonical and the canonical TGFβ pathways (78). After ERK phosphorylation blockade by SC1 (pluripotin), a dual kinase (ERK1, MAPK3) inhibitor (41), we found a significant reduction of C5a-induced SMAD3 activation and of C5a-induced collagen production. In accordance with recent evidences (79,80), these results suggest that ERK might regulate TGF-β/Smad signaling. Therefore, next to TGFβ (81), also innate immune signaling (59) (i.e., anaphylotoxins) might lead to an amplification of interstitial extracellular matrix accumulation by generating myofibroblast via PMT after AKI (82). In accordance with our in vitro findings, the in vivo C5aR1 deficiency protected from PMT, indicating that C5a/C5aR1 is involved in tubulointerstitial fibrosis as shown by Martin et al. (83).
In conclusion, our data suggest that in the early phase of I/R injury, renal pericytes are a major target of complement activation resulting in maladaptive response and PMT. Considering the pivotal role of renal pericytes in preserving vascular homeostasis and maintaining blood perfusion, our data offer new insight into the pathogenic mechanisms regulating vascular capillary reduction and fibrosis development in AKI with potential future therapeutic application.

eThics sTaTeMenT
The study was approved by the ethical committee of the Ministry of Health, Italy. This work was supported by University of Bari   FigUre s1 | Ischemia/reperfusion (I/R) injury did not induce apoptosis or proliferation within perivascular compartment. Quantification of Casp3 + (a) and Ki-67 + (b) cells by IHC after I/R injury with distinction between tubular and perivascular cells. At different times from reperfusion, the Casp3 + and Ki-67 + were detected predominately at tubular level. Results are expressed as mean ± SD of Casp3 + or Ki-67 + cells/high power fields (HPF). Immunofluorescence images showing interstitial peritubular capillaries pericytes co-labeled with PDGFRβ (green) and C3 (red). In T0, PDGFRβ + /C3 + perivascular cells were barely detectable (c). After 30 min of I/R injury [(D), rectangle area was zoomed in (g)], the number of PDGFRβ + /C3 + cells increased predominately at peritubular capillaries level [(D), arrowheads]. C1-INH treatment limited the C3 deposition (e). Isotype control staining was used as negative control (F). Results are expressed as median ± interquartile range of the numbers of PDGFRβ + /C3 + cells/HPF of five independent pigs for each group (h). Magnification, 50×. FigUre s3 | Schematic pathway showing the possible cross-talk between C5aR and TGFβ canonical and non-canonical pathway. C5aR, a G proteincoupled receptor for C5a anaphylotoxin, promotes the MAPK signaling activation (Ras/Raf/MEK), inducing the extracellular signal-regulated kinases (ERK) phosphorylation and transcription of pro-inflammatory and pro-fibrotic genes. pERK activation is also one of the final effector factors of SMADindependent TGFβ pathway (non-canonical pathway) that include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/AKT pathways (not showed). Independently from TGFβ presence, C5a could activate SMAD-independent signaling leading to activation of profibrotic pathway. In addition, pERK could be involved in the activation of SMAD-dependent TGFβ pathway (canonical pathway, green arrow and factors) inducing the SMAD2/3 phosphorylation (red dotted arrow). As common downstream mechanisms, the C5a exposition led to transcription of profibrotic gene and proteinase for detachment. Blocking of pERK, by SC1 (Pluripotin) a dual kinase (ERK1, MAPK3) inhibitor of Thr-202/Tyr-204 phosphorylation could interferes with C5a-induced transcription of proinflammatory and with SMAD3 phosphorylation. This could lead to the decrease of extracellular matrix protein accumulation by perivascular pericytes (C5aR, complement component C5a Receptor 1; MAPK, mitogen-activated protein kinase, ERK, extracellular signal-regulated kinases; SMAD, small mother against decapentaplegic).