Regulation of rheumatoid synovial cell growth by ceramide

Overgrowth of rheumatoid synoviocytes, which results in joint destruction, is due to impaired balance between cell proliferation and cell death (apoptosis). Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. We investigated the effects of ceramide on growth-promoting anti-apoptotic signals in rheumatoid synovial cells. Human synovial cells isolated from patients with rheumatoid arthritis (RA) were stimulated with platelet-derived growth factor (PDGF) in the presence or absence of C2-ceramide. The kinase activity of Akt, MEK, and ERK1/2 was analyzed in PDGF-stimulated synovial cells by Western blot analysis. Pretreatment with C2-ceramide completely inhibited PDGF-induced cell cycle progression of rheumatoid synovial cells. PDGF stimulation induced phosphorylation and activation of Akt, MEK, and ERK1/2 in rheumatoid synovial cells. C2-ceramide inhibited the activation of Akt, MEK and ERK1/2 in PDGF-stimulated synovial cells. Our data demonstrated that inhibition of anti-apoptotic kinases, such as Akt and ERK1/2, may play an important role in ceramide-mediated apoptosis of rheumatoid synovial cells.     

PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived growth factor (PDGF) binding proteins that compete with cell-surface receptors on fibroblasts for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of 15 ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated growth 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on growth or were inhibitory to PDGF-stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Platelet-derived growth factor-stimulated migration of murine fibroblasts is associated with epidermal growth factor receptor expression and tyrosine phosphorylation

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.     

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.     

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.     

Epigallocatechin-3-O-gallate inhibits fibroblast contraction of floating collagen gel: interaction between epigallocatechin-3-O-gallate and platelet derived growth factor

The effects of (-)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts were investigated. EGCG inhibited collagen gel contraction dose-dependently. On the basis of the fact that platelet-derived growth factor (PDGF) is one of the serum components with stimulatory activity in collagen gel contraction, we examined the possibility that interaction between EGCG and PDGF may be involved in this inhibition mechanism. We confirmed this by recombinant PDGF-BB in the present system and we found that EGCG inhibited PDGF-stimulated collagen gel contraction. The results of affinity chromatography indicated that PDGF was bound by EGCG immobilized on agarose gel as detected by enzyme-linked immunoassay and Western blotting. These findings suggest that binding of EGCG to PDGF is at least partly involved in the mechanism of inhibition of collagen gel contraction by EGCG.     

Imatinib mesylate inhibits proliferation and exerts an antifibrotic effect in human breast stroma fibroblasts

Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21(WAF1), leading to the inhibition of fibroblast proliferation, by arresting them at the G(0)/G(1) phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor-mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III-the main collagen types in breast stroma-and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression.     

Atrial natriuretic factor inhibits proliferation of vascular smooth muscle cells stimulated by platelet-derived growth factor

The effect of atrial natriuretic factor (Isoleucine-ANF 101-126) on basal and platelet-derived growth factor (PDGF)-stimulated proliferation of rat aortic vascular smooth muscle cells (VSMC) was assessed by microscopy and measurement of incorporation of tritiated thymidine by cells cultured with or without addition of PDGF in the presence of various concentrations (10(-8)-10(-6) molar) of ANF. ANF had little effect on proliferation of cells grown in media supplemented with 2% fetal calf serum (FCS) alone but exhibited clear dose-related inhibition of PDGF-stimulated thymidine incorporation.     

Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.     

Expression of platelet derived growth factor family members and the potential role of imatinib mesylate for cervical cancer

Background  

Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease; hence newer therapeutical modalities are needed. Preliminary investigations suggest that platelet-derived growth factor (PDGF) might have a role in the development of cervical cancer, therefore it is important to determine whether this growth factor pathway is functional and its targeting with imatinib mesylate leads to growth inhibition of cervical cancer cells.     

Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.     

Progesterone sensitizes breast cancer MCF7 cells to imatinib inhibitory effects

In previous studies, we found that progesterone was able to induce the expression of platelet-derived growth factor (PDGF) in human breast cancer MCF7 cells. Knowing that imatinib mesylate targets PDGF receptor tyrosine kinase activity, the aim of the present study was to examine the effects of imatinib on progesterone-treated MCF7 cells. Expression of phosphorylated (activated) platelet-derived growth factor receptor-alpha (PDGFRalpha) was detected in MCF7 cells. Interestingly, phosphorylated-PDGFRalpha expression was significantly downregulated by imatinib. The effects of imatinib on cell growth, apoptosis and migration were then analyzed. Imatinib effectively inhibited anchorage-dependent colony formation, and cell viability as evaluated by MTT assay. Corroborating these findings, a significant increase in the percentage of apoptotic cells was also observed when cells were treated with imatinib. Surprisingly, these inhibitory effects were all enhanced by the presence of progesterone. Cell migration assays did also show a reduction in the migratory capacity after incubation with imatinib. These findings reveal that imatinib acts by decreasing MCF7 cell viability, growth and migration, with concomitant increase in apoptosis. Furthermore, incubation with progesterone seems to prompt cells to the inhibitory action of imatinib, probably by sustaining PDGFRalpha activity. The current study points out imatinib as a possible therapeutic strategy in progesterone-dependent breast cancer.     

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-beta in mesangial cells

Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet-derived growth factor (PDGF)-stimulated rat mesangial cell mitogenesis. EPA dose-dependently inhibited PDGF-stimulated [(3)H]-thymidine incorporation. PDGF-induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 micro g/ml EPA. Similarly, PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin-dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF-beta secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti-TGF-beta antibody inhibited the EPA-induced suppression of [(3)H]-thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF-beta, did not affect EPA's growth inhibitory effect. These results demonstrate that EPA inhibits PDGF-stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF-beta.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

Apolipoprotein E receptor binding versus heparan sulfate proteoglycan binding in its regulation of smooth muscle cell migration and proliferation

This study showed that synthetic peptides containing either a single copy or tandem repeat of the receptor binding domain sequence of apolipoprotein (apo) E, or a peptide containing its C-terminal heparin binding domain, apoE-(211-243), were all effective inhibitors of platelet-derived growth factor (PDGF)-stimulated smooth muscle cell proliferation. In contrast, only the peptide containing a tandem repeating unit of the receptor binding domain sequence of apoE, apoE-(141-155)(2), was capable of inhibiting PDGF-directed smooth muscle cell migration. Peptide containing only a single unit of this sequence, apoE-(141-155), or the apoE-(211-243) peptide were ineffective in inhibiting PDGF-directed smooth muscle cell migration. Additional experiments showed that reductively methylated apoE, which is incapable of receptor binding yet retains its heparin binding capability, was equally effective as apoE in inhibiting PDGF-stimulated smooth muscle cell proliferation. However, reductively methylated apoE was unable to inhibit smooth muscle cell migration toward PDGF. Additionally, the receptor binding domain-specific apoE antibody 1D7 also mitigated the anti-migratory properties of apoE on smooth muscle cells. Finally, pretreatment of cells with heparinase failed to abolish apoE inhibition of smooth muscle cell migration. Taken together, these data documented that apoE inhibition of PDGF-stimulated smooth muscle cell proliferation is mediated by its binding to heparan sulfate proteoglycans, while its inhibition of cell migration is mediated through apoE binding to cell surface receptors.     

Serine phosphorylation of the ligand-activated beta-platelet-derived growth factor receptor by casein kinase I-gamma2 inhibits the receptor's autophosphorylating activity.

Platelet-derived growth factor (PDGF) receptors (PDGFRs) are membrane protein-tyrosine kinases that, upon activation, become tyrosine-phosphorylated and associate with numerous SH2 domain-containing molecules involved in mediating signal transduction. In Rat-2 fibroblasts, we have characterized the phosphorylation of the beta-PDGFR following its activation by PDGF. In contrast to tyrosine phosphorylation, which was transient and returned to near basal levels by 30 min, PDGF-stimulated Ser/Thr phosphorylation of the beta-PDGFR was increased by 5 min and remained elevated after 30 min. In vivo, after 5 min of PDGF stimulation, serine phosphorylation of the beta-PDGFR was greatly reduced by CKI-7, a specific inhibitor of casein kinase I (CKI). In vitro, recombinant CKI-gamma2 phosphorylated the ligand-activated beta-PDGFR on serine residues in a CKI-7-sensitive manner and resulted in a marked inhibition of the receptor's autophosphorylating activity. Furthermore, in Rat-2 fibroblasts, expression of hemagglutinin epitope-tagged active CKI-gamma2 resulted in a dramatic decrease in the tyrosine phosphorylation state of the beta-PDGFR in response to PDGF, consistent with receptor inactivation. Our data suggest that upon PDGF stimulation, CKI-gamma2 is activated and/or translocated in proximity to the beta-PDGFR, whereby it phosphorylates the beta-PDGFR on serine residues and negatively regulates its tyrosine kinase activity, leading to receptor inactivation.     

Epoxyeicosatrienoic acids induce growth inhibition and calpain/caspase-12 dependent apoptosis in PDGF cultured 3T6 fibroblast

Previous studies have demonstrated that arachidonic acid (AA) metabolites released by the cyclooxygenase pathway is involved in serum-induced 3T6 fibroblast cycle progression and proliferation. However, these results also suggest that other AA cascade pathways might be involved. Recently, we also described the role of hydroxyeicosatetraenoic acids, which are produced by cytochrome P450 monooxygenases (CYP), in 3T6 fibroblast growth. AA can be also metabolized by the epoxygenase activity of CYP-producing epoxyeicosatrienoic acids (EETs). Finally, the cytosolic epoxide hydrolases catalyze the hydration of the EETs, transforming them into dihydroxyeicosatetraenoic acids (DHETEs). In this work, we have studied the role of the EETs/DHETEs on 3T6 fibroblasts growth. Our results show that PDGF stimulates 3T6 fibroblast proliferation and [³H]thymidine incorporation, while the addition of 5,6-EET, 8,9-EET, 11,12-EET or 14,15-EET (0.1–1 μM) inhibit these processes. Furthermore, 5,6-DHETE and 11,12-DHETE (0.1–1 μM) also inhibit cell proliferation and DNA synthesis. Interestingly, this growth inhibition was correlated with an induction of apoptosis. Thus, we observed that in the presence of PDGF, EETs or DHETEs (0.1–1 μM) induce phosphatidylserine externalization (as measured by annexin V-binding) and DNA fragmentation (as quantified using a TUNEL assay). Our results show that calpain, as well as caspase-12 and caspase-3, are involved in these events. Therefore, EETs and DHETEs have anti-proliferative and pro-apoptotic effects on PDGF-stimulated 3T6 fibroblasts.