Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

: Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.

Echocardiographic evaluation of four giant Aldabra tortoises (Aldabrachelys gigantea).

OBJECTIVES: In recent years echocardiography has become a good diagnostic tool in Zoo Medicine but in some cases, it is still a challenge. In Aldabra giant tortoise (Aldabrachelys gigantea) the big size of animals and the few individuals hosted in Zoo are critical points for the application of this diagnostic technique.The purposes of this research were: to evaluate the feasibility of the diagnostic imaging technique on big-sized turtles; to define the echographic parameters for this species; and to describe the morphofunctional and physiological echographic characteristics of their cardiovascular system. DESIGN: Repeated measures in vivo. SETTING: Ultrasonography systematic description and Doppler analysis of the cardiovascular system of Aldabra giant tortoise were carried out; B-mode examination allowed the evaluation of the kinetics of the ventricle, the atria and the atrioventricular valves. PARTICIPANTS: 4 Aldabra giant tortoises (two adult males and two young females) hosted in two zoological gardens. INTERVENTIONS: Echocardiography was performed placing the animals in ventral on a restraining platform raised from the floor, to provide adequate accessibility to the thoracic windows where the probe was placed. No chemical restraint was used. PRIMARY AND SECONDARY OUTCOME MEASURES: Heart rate, systolic and diastolic areas and volumes, vessel diameters and blood flow velocity were measured. RESULTS: Heart rate was 21±4 bpm (range 14-25 bpm). The averages of the diastolic and systolic area indexes linked to the subject weight were: 21±3 cm2 and 9±1 cm2.The aortic annulus diameter in female specimens measured 11.2±0.8 mm, while it measured 21.5±0.3 mm in male species. CONCLUSION: Results confirm the effectiveness of echocardiography as a means to study and evaluate the cardiovascular system of this species even if more studies on a bigger number of patients would be necessary to develop the echocardiography technique.

15-deoxy-delta 12,14-prostaglandin J(2) inhibits the synthesis of the acute phase protein SIP24 in cartilage: Involvement of COX-2 in resolution of inflammation.

We previously demonstrated that, in the MC615 cartilage cell line, the p38/NF-kB pathway is activated both during differentiation and in response to an inflammatory stimulus. In both cases, the p38/NF-kB pathway activation leads to the expression of the lipocalin SIP24 and of COX-2. Given the fact that, in the same cells, the COX-2 expression is sustained during the inflammation resolution, at the same time that the SIP24 expression is suppressed, in the present study we tested the hypothesis that COX-2 products play a role in SIP24 repression. Taken together, our results suggest that, during the resolution of inflammation, COX-2 represses the acute phase protein SIP24 and restores physiological conditions, possibly through a pathway involving PPARgamma. Experimental evidences being the following: (1) 15-deoxy-delta 12,14-prostaglandin J(2), but not PGE(2): (i) inhibits the expression of SIP24 in the inflammatory phase and induces COX-2 synthesis; (ii) represses NF-kB activation induced by LPS; (iii) represses the synthesis of microsomal PGE Synthase-1 induced by LPS. (2) PPARgamma and PPARalpha are present in MC615 cells in both proliferating and hyperconfluent cultures. (3) PPARgamma ligand GW7845, but not PPARalpha ligand GW7647: (i) represses the expression of SIP24 induced by LPS; (ii) induces COX-2 expression. (4) p38 is involved in the PPARgamma mediated induction of COX-2. In fact 15-deoxy-delta 12,14-prostaglandin J(2) activates p38 and the cell pretreatment with the p38 specific inhibitor SB203580 represses the expression of COX-2 induced by both the 15-deoxy-delta12,14-prostaglandin J(2) and the PPARgamma ligand GW7845.

p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes.

Studying cartilage differentiation, we observed the emergence of inflammation-related proteins suggesting that a common pathway was activated in cartilage differentiation and inflammation. In the present paper, we investigated the expression pathway of the inflammation-related enzyme Cyclooxygenase-2 (COX-2) during differentiation and inflammatory response of the chondrocytic cell line MC615. Cells were cultured either as (i) proliferating prechondrogenic cells expressing type I collagen or (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. The p38 and the NF-kB pathways were investigated in standard conditions and after inflammatory agents treatment. NF-kB was constitutively activated in differentiated cells. The activation level of NF-kB in differentiated cells was comparable to the level in proliferating cells treated with the inflammatory agent LPS. In both cases, p65 was bound to the NF-kB consensus sequence of COX-2 promoter. p38, constitutively activated in differentiated cells, was activated in proliferating cells by treatment with LPS or IL-1alpha. In stimulated proliferating cells the two pathways are connected since addition of the p38-specific inhibitor SB203580 inhibited p38 activation, significantly reduced NF-kB activation and repressed COX-2 synthesis indicating that p38 is upstream NF-kB activation and COX-2 synthesis. In differentiated cells, the treatment with the inflammatory agent neither enhance NF-kB activation, nor synthesis of COX-2 while the addition of SB203580 neither repressed activation of p38, nor COX-2 synthesis, suggesting a constitutive activation of a p38/NF-kB/COX2 pathway. Our data indicate that in chondrocytes, COX-2 is expressed via p38 activation/NF-kB recruitment during both differentiation and inflammatory response.

Multiple Organ Dysfunction Syndrome (MODS) induced by Candida krusei in an Aldabra giant tortoise (Aldabrachelys gigantea) and confirmed by electron microscopy analysis.

A young female Aldabra giant tortoise (Adabrachelys gigantea) was presented with anorexia, ataxia, severe constipation and bloating. Analysis revealed liver disease and collected biopsy diagnosed Candida krusei infection. Despite Itraconazole treatment, the tortoise got worse and died. Full necropsy was performed; microbiology showed Candida krusei presence in liver, but histopathology didn't confirm fungal presence with special stains, so scanning electron microscopy was essential to prove a detailed diagnosis of extensive mycosis.

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo ). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018-1028.

Expression of serum amyloid A in chondrocytes and myoblasts differentiation and inflammation: possible role in cholesterol homeostasis.

Serum amyloid A (SAA) is synthesized by the liver during the acute phase. Local expression of SAA mRNA has been reported also in non-liver cells, a potential local source of SAA protein not related to the systemic acute phase response. SAA function has not been established yet. In the present study, we identified SAA as a protein expressed by chondrocytes and myoblasts in response to inflammatory stimula. In both cell systems, SAA mRNA and protein expression is strongly stimulated by bacterial lipopolysaccharide treatment. SAA mRNA expression is also enhanced during terminal differentiation of cells of the chondrogenic and myogenic lineage; mRNA is barely detectable in prechondrogenic cells and is highly expressed in differentiated hyperthrophic chondrocytes. An increased level of SAA mRNA was also observed in vivo when we compared mRNA extracted from tibiae of 10 day embryos, still fully cartilaginous, with tibiae from 18 day embryos, a stage when the endochondral ossification process has already started. p38 activation, a well-known event of the chondrogenesis signaling cascade, controls expression of SAA in cartilage following inflammatory stimuli. SAA secreted by stimulated chondrocytes is associated with cholesterol. Cholesterol is synthesized by the same chondrocytes and is also increased in inflammatory conditions. A role of SAA in cholesterol homeostasis in chondrocytes is proposed.

Mesenchymal stem cell paracrine activity is modulated by platelet lysate: induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype.

Wound healing is achieved through distinct programmed phases: hemostasis, inflammation, mesenchymal cell proliferation and migration, and tissue remodeling. At the injury site, clot formation and platelet degranulation release cytokines and growth factors and actively participating in the healing process and regulating the migration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes. We previously demonstrated that, in an inflammatory environment, prostaglandin E2 (PGE2) secreted by mesenchymal stem cells (MSCs) promoted the macrophage switch from a proinflammatory to a proresolving phenotype. Using an in vitro model, we here evaluated the role carried out by the two main players of the wound healing process, the platelet degranulation content mimicked by the platelet lysate (PL) and the inflammatory stimulus, on the modulation of mouse bone-marrow-derived MSC paracrine activity. We demonstrated that, in MSCs, PL induced nuclear factor kappaB (NF-κB) activation, expression of COX-2 and mPGE synthase, and PGE2 production; in an inflammatory microenvironment, PL increased the inflammatory response and promoted the secretion of the proinflammatory cytokine IL-6. We assayed on mouse primary macrophages the paracrine activity of MSCs exposed to the different microenvironments and we observed that PL-treated MSC-conditioned medium maintained macrophages in a proinflammatory state. The involved factors were granulocyte macrophage-colony stimulating factor induced by PL in MSCs and TNF-α induced by PL-MSC-conditioned medium in macrophages. Our findings indicate that PL triggers an inflammatory response in MSCs and induces the secretion of factors maintaining macrophages in a proinflammatory state thus enhancing the initial inflammatory response to the injury, a key element in the activation of wound healing.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair.

In vivo implanted bone marrow-derived mesenchymal stem cells trigger a cascade of cellular events leading to the formation of an ectopic bone regenerative niche.

We recently reported that mouse bone marrow stromal cells, also known as bone marrow (BM)-derived mesenchymal stem cells (MSCs), seeded onto a scaffold and implanted in vivo, led to an ectopic bone deposition by host cells. This MSCs capacity was critically dependent on their commitment level, being present only in MSCs cultured in presence of fibroblast growth factor-2. Taking advantage of a chimeric mouse model, in this study we show that seeded MSCs trigger a cascade of events resulting in the mobilization of macrophages, the induction of their functional switch from a proinflammatory to a proresolving phenotype, and the subsequent formation of a bone regenerative niche through the recruitment, within the first 2 weeks of implantation, of endothelial progenitors and of cells with an osteogenic potential (CD146+CD105+), both of them derived from the BM. Moreover, we demonstrated that, in an inflammatory environment, MSCs secrete a large amount of prostaglandin E2 playing a key role in the macrophage phenotype switch.

Platelet-rich plasma exerts antinociceptive activity by a peripheral endocannabinoid-related mechanism.

In regenerative medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent antinociceptive effect against both early and late nocifensive responses. This effect was observed following intrapaw injection of (1) total PRP; (2) lipids extracted from PRP; and (3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB2 receptors, injected in the paw, abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent antinociceptive activity linked, at least in part, to their endocannabinoids and related compound content, and to their capability of elevating the levels of these lipid mediators in cells.

Platelet lysate induces in vitro wound healing of human keratinocytes associated with a strong proinflammatory response.

Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro: activation of an anti-inflammatory cyclooxygenase-2 (COX-2) pathway.

INTRODUCTION: The mono- and digalactosyldiacylglycerol (MGDG and DGDG) galactolipids have been purified from the thermophilic blue-green alga Phormidium sp. ETS-05 that colonizes the therapeutic thermal mud of Abano Terme and Montegrotto Terme, Italy. Both compounds present a marked composition in polyunsaturated fatty acids, mainly omega-3. The therapeutic thermal mud is applied mainly to osteoarthritic cartilage patients. In the present study the effect of MGDG treatment on proteins and factors expressed by human articular cartilage cells in culture and on pathways activated in inflammatory conditions was studied. METHODS: Primary cultures of human articular chondrocytes were used at cell passage number 1 (P1). Cells were treated in serum-free medium with inflammatory cytokines in the presence and in the absence of MGDG. Western blot was performed on collected medium and on cell layers. At least three different experiments were performed on primary cultures. The quantitation of the MGDG effect was performed by densitometric scanning of Western blots. p38 Mitogen Activated Protein Kinase (p38) activation, Nuclear Factor-kappaB (NF-kB) activation and Prostaglandin E(2) (PGE(2)) quantitation were performed by commercially available assays. Results are given as the mean values ± SD. All statistical analyses were performed using GraphPad software. The two-tailed Student's t -test was performed. RESULTS: We report that MGDG: 1) represses the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) induced by interleukin-1alpha (IL-1α) or IL-1α + tumor necrosis factor α (TNFα) interfering with the p38 and NF-kB pathways; 2) is not toxic for the cells and does not affect the cell phenotype; 3) strongly enhances COX-2 expression induced by IL-1α or IL-1α + TNFα; 4) represses mPGES expression induced by IL-1α and the synthesis of PGE(2) and induces the synthesis of 15-deoxy-Δ 12,14-prostaglandin J(2) (15ΔPGJ(2)). In addition, the COX-2 product 15ΔPGJ(2) added to the cells: 1) strongly represses IL-6 and IL-8 induced by IL-1α; 2) represses mPGES expression induced by IL-1α and the synthesis of PGE(2). CONCLUSIONS: All together these data suggest that MGDG has an anti-inflammatory activity in human articular cartilage and possibly activates an anti-inflammatory loop triggered by COX-2 via 15ΔPGJ(2) production, indicating a possible role of COX-2 in resolution of inflammation. The purified compound is a novel anti-inflammatory agent potentially active for human articular cartilage pathologies related to inflammation.

Role of MT1-MMP in the osteogenic differentiation.

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.

A common pathway in differentiation and inflammation: p38 mediates expression of the acute phase SIP24 iron binding lipocalin in chondrocytes.

SIP24 is an acute phase iron binding lipocalin physiologically expressed in vivo in developing cartilage by prehypertrophic/hypertrophic chondrocytes. Taking advantage of the chondrocytic cell line MC615 and using SIP24 as a marker we investigated the pathways active in cartilage differentiation and inflammation. MC615 cells were cultured as: (i) proliferating prechondrogenic cells expressing type I collagen (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. In proliferating cells the pathway PKC/ERK1, ERK2 was activated and SIP24 was not expressed while in differentiated cells the pathway p38/NF-kappaB was activated and SIP24 was expressed. Proliferating cells treated with inflammatory agents expressed a large amount of SIP24 and showed activation of p38/NF-kappaB pathway and inhibition of PKC/ERK1, ERK2 pathway indicating that in inflammation and differentiation the same factors are activated (p38, NF-kappaB) or inactivated (PKC, ERKs). Treatment of proliferating cells with the p38 specific inhibitor SB203580 inhibited the inflammation induced activation of p38 and the synthesis of SIP24. PMA treatment induced activation of PKC, inactivation of p38 and suppression of SIP24 synthesis, suggesting that PKC activation inhibits p38 activation. In differentiated hyperconfluent cells the same factors (p38/NF-kappaB/SIP24) are constitutively activated: treatment with inflammatory agents does not increase synthesis of SIP24 while treatment with SB203580 and with PMA does not repress activation of p38 nor synthesis of SIP24. We propose that the SIP24 stress related protein is expressed via p38 activation/NF-kappaB recruitment both in chondrocyte differentiation and inflammation and that a signaling pathway active in the acute phase response is physiologically activated in differentiation.

Pro-collagen I COOH-terminal trimer induces directional migration and metalloproteinases in breast cancer cells.

Breast and prostatic carcinomas, melanoma, and endothelial cell lines are chemoattracted by medium conditioned by mature osteoblasts. The chemoattractant for endothelial cells was identified with C3, carboxyl-terminal trimer of pro-collagen type I. We report that C3 induces directional migration and proliferation, the expression of tissue inhibitor of metalloproteinases-2, pro-metalloproteinase-2 and -9, and their activation in MDA MB231 cells, without changing the expression of tissue inhibitor of metalloproteinases-1 and of metalloproteinase-14. Antiserum against metalloproteinase-2 or -9 or -14, tissue inhibitor of metalloproteinases-1, or GM6001 inhibits the C3-induced migration. Urokinase and its receptor are detected and unchanged upon exposure to C3. The antibody against urokinase or addition of plasminogen activator inhibitor inhibits migration. Blocking antibodies to integrins alpha(2), alpha(6), beta(1), and beta(3) inhibit chemotaxis and do not change urokinase and urokinase receptor expression. Blockage of alpha(2), beta(1), and beta(3) integrins affect differently the induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits C3-induced pro-metalloproteinase -2 and -9, but not urokinase expression. Wortmannin partially inhibits C3-induced cell migration. Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the only agent known to induce migration specifically of both endothelial and breast carcinoma cells. The mitogenic and motogenic role of C3 in vitro might prefigure a role in in vivo carcinogenesis and in the establishment of metastasis.