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Chemical linkages that respond to biological stimuli are important for many pharmaceutical and biotechnological applications, making it relevant to explore new variants with different responsivity profiles. This work explores the responsiveness of a TAT peptide-based sulfonium vinyl sulfide probe that responds to nucleophilic thiols, radical thiol species (RTS), and reactive nitrogen species (RNS). Under model conditions, response to nucleophilic thiols was very slow (hours/days), though fast with down to molar equivalents of either RTS or RNS (minutes). These reactions led to the traceless release of a methionine-containing peptide in the first two cases and to a hydroxy nitration adduct in the third case. Despite the sensitive nature of the probe, it remained stable for at least â¼2 h in the presence of cells during TAT-mediated trafficking, even under pro-inflammatory stimulation. The thiol-responsiveness is intermediate to that observed for disulfide linkers and conventional cysteine-maleimide linkers, presenting opportunities for biotechnological applications.
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OBJECTIVE: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. DESIGN: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. RESULTS: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. CONCLUSION: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.
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Neoplasias Colorretais , Microbioma Gastrointestinal , Camundongos , Animais , Citocinas , Microbioma Gastrointestinal/fisiologia , Estudos Retrospectivos , RNA Ribossômico 16S , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica/microbiologia , Neoplasias Colorretais/cirurgiaRESUMO
Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.
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Antígenos de Superfície , Proteínas do Leite , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Aorta/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Fator VIII , Glicolipídeos , Glicoproteínas , Gotículas Lipídicas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Leite/genética , Proteínas do Leite/metabolismoRESUMO
BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a catastrophic disease with devastating consequences, including a high mortality rate and severe disabilities among survivors. Inflammation is induced following SAH, but the exact role and phenotype of innate immune cells remain poorly characterized. We investigated the inflammatory components of the early brain injury in an animal model and in SAH patients. METHOD: SAH was induced through injection of blood in the subarachnoid space of C57Bl/6 J wild-type mice. Prospective blood collections were obtained at 12 h, days 1, 2, and 7 to evaluate the systemic inflammatory consequences of SAH by flow cytometry and enzyme-linked immunosorbent-assay (ELISA). Brains were collected, enzymatically digested, or fixed to characterize infiltrating inflammatory cells and neuronal death using flow cytometry and immunofluorescence. Phenotypic evaluation was performed at day 7 using the holding time and footprint tests. We then compared the identified inflammatory proteins to the profiles obtained from the plasma of 13 human SAH patients. RESULTS: Following SAH, systemic IL-6 levels increased rapidly, whereas IL-10 levels were reduced. Neutrophils were increased both in the brain and in the blood reflecting local and peripheral inflammation following SAH. More intracerebral pro-inflammatory monocytes were found at early time points. Astrocyte and microglia activation were also increased, and mice had severe motor deficits, which were associated with an increase in the percentage of caspase-3-positive apoptotic neurons. Similarly, we found that IL-6 levels in patients were rapidly increased following SAH. ICAM-1, bFGF, IL-7, IL-12p40, and MCP-4 variations over time were different between SAH patients with good versus bad outcomes. Moreover, high levels of Flt-1 and VEGF at admission were associated with worse outcomes. CONCLUSION: SAH induces an early intracerebral infiltration and peripheral activation of innate immune cells. Furthermore, microglia and astrocytic activation are present at later time points. Our human and mouse data illustrate that SAH is a systemic inflammatory disease and that immune cells represent potential therapeutic targets to help this population of patients in need of new treatments.
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Encéfalo/imunologia , Encéfalo/patologia , Imunidade Inata/fisiologia , Hemorragia Subaracnóidea/imunologia , Hemorragia Subaracnóidea/patologia , Animais , Encéfalo/metabolismo , Lesões Encefálicas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hemorragia Subaracnóidea/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein ß2-glycoprotein I (ß2GPI). Notably, mice immunized with ß2GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited ß2GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that ß2GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their ß2GPI T cell epitope specificity or MHC class II haplotype. Although ß2GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different ß2GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one ß2GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403(+) autoimmune patient. These data suggest that the generation of a ß2GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a ß2GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.
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Autoanticorpos/imunologia , Epitopos de Linfócito T/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T/imunologia , beta 2-Glicoproteína I/imunologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/metabolismo , Feminino , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Haplótipos/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Hibridomas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Linfócitos T/metabolismo , beta 2-Glicoproteína I/metabolismoRESUMO
ß(2)-Glycoprotein I (ß(2)GPI) is an abundant plasma protein that binds to the surface of cells and particles expressing negatively charged lipids, but its physiological role remains unknown. Antibodies to ß(2)GPI are found in patients with anti-phospholipid syndrome, a systemic autoimmune disease associated with vascular thrombosis and pregnancy morbidity. Although it has been suggested that anti-ß(2)GPI antibodies activate endothelial cells and monocytes by signaling through TLR4, it is unclear how anti-ß(2)GPI antibodies and/or ß(2)GPI interact with TLR4. A number of mammalian proteins (termed "endogenous Toll-like receptor (TLR) ligands") have been reported to bind to TLR4, but, in most cases, subsequent studies have shown that LPS interaction with these proteins is responsible for TLR activation. We hypothesized that, like other endogenous TLR ligands, ß(2)GPI interacts specifically with LPS and that this interaction is responsible for apparent TLR4 activation by ß(2)GPI. Here, we show that both LPS and TLR4 are required for ß(2)GPI to bind to and activate macrophages. Untreated ß(2)GPI stimulated TNF-α production in TLR4-sufficient (but not TLR4-deficient) macrophages. In contrast, neither polymyxin B-treated nor delipidated ß(2)GPI stimulated TNF-α production. Furthermore, ß(2)GPI bound to LPS in a specific and dose-dependent manner. Finally, untreated ß(2)GPI bound to the surface of TLR4-sufficient (but not TLR4-deficient) macrophages. Polymyxin B treatment of ß(2)GPI abolished macrophage binding. Our findings suggest a potential new biological activity for ß(2)GPI as a protein that interacts specifically with LPS and point to the need to evaluate newly discovered endogenous TLR ligands for potential interactions with LPS.
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Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , beta 2-Glicoproteína I/metabolismo , Animais , Endotoxinas/metabolismo , Feminino , Regulação da Expressão Gênica , Imunidade Inata , Ligantes , Lipídeos/química , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Polimixina B/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Milk fat globule-epidermal growth factor-8 (MFG-E8) is a glycoprotein secreted by different cell types, including apoptotic cells and activated macrophages. MFG-E8 is highly expressed in a variety of cancers and is classically associated with tumor growth and poor patient prognosis through reprogramming of macrophages into the pro-tumoral/pro-angiogenic M2 phenotype. To date, correlations between levels of MFG-E8 and patient survival in prostate and renal cancers remain unclear. Here, we quantified MFG-E8 and CD68/CD206 expression by immunofluorescence staining in tissue microarrays constructed from renal (n = 190) and prostate (n = 274) cancer patient specimens. Percentages of MFG-E8-positive surface area were assessed in each patient core and Kaplan-Meier analyses were performed accordingly. We found that MFG-E8 was expressed more abundantly in malignant regions of prostate tissue and papillary renal cell carcinoma but was also increased in the normal adjacent regions in clear cell renal carcinoma. In addition, M2 tumor-associated macrophage staining was increased in the normal adjacent tissues compared to the malignant areas in renal cancer patients. Overall, high tissue expression of MFG-E8 was associated with less disease progression and better survival in prostate and renal cancer patients. Our observations provide new insights into tumoral MFG-E8 content and macrophage reprogramming in cancer.
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Complications following endovascular aneurysm repair (EVAR) are related to deficient healing around the stent-graft (SG). New generations of SG with surface properties that foster vascular repair could overcome this limitation. Our goal was to evaluate the potential of a new nitrogen-rich plasma-polymerised biomaterial, designated PPE:N, as an external coating for polyethylene terephtalate (PET)- or polytetrafluoro-ethylene (PTFE)-based SGs, to promote healing around the implant. Thin PPE:N coatings were deposited on PET and PTFE films. Then, adhesion, growth, migration and resistance to apoptosis of vascular smooth muscle cells (VSMCs) and fibroblasts, as well as myofibroblast differentiation, were assessed in vitro. In another experimental group, chondroitin sulphate (CS), a newly described mediator of vascular repair, was added to normal culture medium, to search for possible additional benefit. PPE:N-coatings, especially on PET, increased and accelerated cell adhesion and growth, compared with control PET and with standard polystyrene culture plates (PCP). PPE:N was also found to increase the resistance to apoptosis in VSMC, an important finding as aneurysms are characterised by VMSC depletion caused by a pro-apoptotic phenotype. Addition of CS in solution further increased migration and resistance to apoptosis. In conclusion, PPE:N-coating and/or CS could promote vascular repair around SGs following EVAR.
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Aneurisma/cirurgia , Prótese Vascular/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Nitrogênio/administração & dosagem , Stents/efeitos adversos , Cicatrização/efeitos dos fármacos , Aneurisma/complicações , Aneurisma/patologia , Animais , Linhagem Celular , Materiais Revestidos Biocompatíveis/administração & dosagem , Materiais Revestidos Biocompatíveis/química , Implantes de Medicamento , Células Endoteliais/patologia , Teste de Materiais , Nitrogênio/química , RatosRESUMO
Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure.
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Antígenos de Superfície/metabolismo , Apoptose/fisiologia , Proteínas do Leite/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Cicatrização/fisiologia , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Distribuição Aleatória , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Mediators released by apoptotic renal resident cells play a crucial role in modification of the inflammatory microenvironment. We have demonstrated that milk fat globule epidermal growth factor 8 (MFG-E8) is released by apoptotic cells, which results in reduced proinflammatory cytokine production by macrophages. The present study was designed to study the role of MFG-E8 on the modulation of tissue damage and macrophage phenotype in a renal inflammatory model, unilateral ureteral obstruction (UUO). C57BL/6 WT or MFG-E8 KO mice underwent ureteral ligation for 3, 7, and 14 d to evaluate renal injury. MFG-E8 (30 µg/kg) or vehicle was also administered i.p. MFG-E8 administration reduced kidney damage and fibrosis compared with control, whereas its absence in MFG-E8 KO mice was associated with more severe disease. Moreover, MFG-E8 administration was associated with decreased inflammasome activation in the kidney. Furthermore, adoptive transfer of MFG-E8-stimulated macrophages reduced activation of inflammasome and tissue damage. In all cases, both the systemic administration of MFG-E8 and MFG-E8-treated macrophages promoted accumulation of anti-inflammatory CD206+ macrophages. We propose that the protective role of MFG-E8 is mediated through anti-inflammatory macrophage reprogramming which results in decreased inflammasome activation, preventing severe tissue damage. These data provide valuable insight for identification of MFG-E8 as a novel target in modulation of inflammatory diseases.
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Antígenos de Superfície/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Proteínas do Leite/imunologia , Nefrite/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/uso terapêutico , Apoptose , Microambiente Celular , Colágeno/análise , Imunoterapia Adotiva , Inflamassomos/efeitos dos fármacos , Rim/química , Rim/imunologia , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Leite/genética , Proteínas do Leite/uso terapêutico , Nefrite/etiologia , Nefrite/patologia , Nefrite/terapia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Obstrução Ureteral/complicaçõesRESUMO
Increased endothelial apoptosis and decreased apoptosis of vascular smooth muscle cells (VSMC) are central to initiation of myo-intimal thickening. We hypothesized that apoptosis of endothelial cells (EC) induces the release of anti-apoptotic mediator(s) active on VSMC. We found that serum-free medium conditioned by apoptotic EC decreases apoptosis of VSMC compared with fresh serum-free medium. Inhibition of endothelial apoptosis during conditioning with a pan-caspase inhibitor ZVAD-FMK blocked the release of the anti-apoptotic factor(s) active on VSMC. VSMC exposed to serum-free medium conditioned by apoptotic EC showed increased ERK 1/2 phosphorylation, enhanced Bcl-xl expression, and inhibition of p53 expression. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as a C-terminal fragment of the domain V of perlecan. Serum-free medium supplemented with either a synthetic peptide containing the EGF motif of the domain V of perlecan or chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, increased ERK 1/2 phosphorylation and Bcl-xl protein levels while inhibiting apoptosis of VSMC. These results suggest that a proteolytic activity developing downstream of activated caspases in apoptotic EC initiates degradation of pericellular proteoglycans and liberation of bioactive fragments with a robust impact on inhibition of VSMC apoptosis.
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Apoptose , Caspases/metabolismo , Endotélio Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Comunicação Parácrina , Animais , Fatores Biológicos/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/fisiologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-XRESUMO
Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components.
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Apoptose/fisiologia , Caspase 3/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Catepsina L , Linhagem Celular , Modelos Animais de Doenças , Humanos , Comunicação Parácrina/fisiologia , Peptídeos/metabolismo , RNA Interferente PequenoRESUMO
Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing alpha2beta1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src(-/-)Fyn(-/-) fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an alpha2beta1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.
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Apoptose , Fibrose/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteoglicanas de Heparan Sulfato/química , Integrina alfa2beta1/química , Quinases da Família src/química , Animais , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Interferência de RNARESUMO
Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-x(L) overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-x(L) protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of alpha-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.