Biomedical subjects

Shigeki Higashiyama

Publications and source records attributed to Shigeki Higashiyama.

At least 37 records · Page 2Linked to original sources

PubMed2004

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

ADAM Proteins↗

PubMed2004

Roles of charged amino acid residues in the cytoplasmic domain of proHB-EGF.

Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane precursor (proHB-EGF). Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C, respectively). We have previously shown that HB-EGF-C is translocated from the plasma membrane into the nucleus, where it interacts with the transcription repressor, PLZF. Here we characterize the amino acid residues of the cytoplasmic domain of proHB-EGF on cell surface distribution and the interaction of HB-EGF-C with PLZF. The cytoplasmic domain contains three characteristic clusters with charged amino acids. Generation of various mutants of proHB-EGF showed that the arrangement of the charged amino acids in the cytoplasmic domain regulates the distribution of proHB-EGF at the plasma membrane but does not regulate proHB-EGF processing and internalization of HB-EGF-C. Further, the charged amino acids are also required for HB-EGF-C-PLZF interaction. These results indicate that the cytoplasmic domain of proHB-EGF is a multifunctional domain.

Amino Acid Motifs↗

PubMed2004

CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells.

CD9, a member of the tetraspanin family, has been shown to be involved in a range of cellular activities, including migration, proliferation and adhesion, but the molecular mechanisms by which it mediates such events is unclear. Here, we found that anti-CD9 monoclonal antibody ALB6 inhibited cell proliferation, reduced cell viability and induced not only morphological changes specific to apoptosis but also molecular changes, as evidenced by TUNEL and annexin-V staining. For the possible mechanism of ALB6-induced apoptosis, ALB6 activated the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 mitogen-activated-protein kinase (MAPK) within 5-15 minutes, as well as caspase-3 within 24-48 hours. It is noteworthy that ALB6 induced tyrosine phosphorylation of the p46 Shc isoform specifically and that the overexpression of its dominant-negative form completely suppressed the ALB6-induced activation of JNK/SAPK, p38 MAPK and caspase-3, resulting in the inhibition of apoptotic cell death. These results suggest that CD9 might regulate apoptosis through the specialized signals in human cancer cell lines.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis.

The "a disintegrin and metalloprotease" (ADAM) family contributes to regulation of the cell-cell and cell-matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin alpha 3 and beta 1 subunits. Administration of the anti-beta 1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.

ADAM Proteins↗

PubMed2004

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands.

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

ADAM Proteins↗

PubMed2004

The N-terminal region of NTAK/neuregulin-2 isoforms has an inhibitory activity on angiogenesis.

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2, is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Because ErbB signaling has been implicated in various angiogenic mechanisms, the effect of NTAK (which has at least nine isoforms due to alternative splicing) in angiogenesis is explored. One isoform, NTAKgamma, inhibited cell growth in terms of DNA synthesis and cell numbers in vascular endothelial cells specifically, whereas NTAKalpha and beta had no activity. On the other hand, NTAKgamma secreted by transfected MDA-MB-231 cells inhibited endothelial cell growth, and NTAKgamma expressed in endothelial cells by adenovirus infection suppressed cell growth in a dose-dependent manner. The EGF-like domain of NTAKgamma did not have this activity. The NTAKdelta isoform, which had the Ig-like domain but not the EGF-like domain, inhibited proliferation of endothelial cells. NTAKdelta prevented hyper-phosphorylation of the retinoblastoma tumor suppressor protein and caused G(1) arrest in endothelial cells. Both NTAKgamma and delta isoforms displayed anti-angiogenic activity in the chick embryo chorioallantoic membrane in vivo. These results suggest that the active site of NTAK is localized outside of the EGF-like domain but within the N-terminal region, including the Ig-like domain, of NTAK.

Amino Acid Sequence↗

PubMed2004

Dual intracellular signaling by proteolytic cleavage of membrane-anchored heparin-binding EGF-like growth factor.

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a membrane-anchored precursor (proHB-EGF) that is cleaved to release a soluble HB-EGF by specific metalloproteases. Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C). Recent studies indicate that the processing of proHB-EGF is strictly regulated and involved in a variety of biological processes and that not only HB-EGF but also HB-EGF-C functions as a signaling molecule. ProHB-EGF generates dual intracellular signaling molecules by its proteolytic cleavage.

Animals↗

PubMed2004

The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells.

BACKGROUND & AIMS: The epidermal growth factor (EGF) receptor (EGFR) can be transactivated by many factors including G-protein-coupled receptor agonists and cytokines. Although this EGFR transactivation reportedly requires a disintegrin and metalloproteinase (ADAM) that sheds the ectodomain of EGFR ligands, the detailed mechanisms are still unknown. This study evaluated the mechanism of interleukin (IL)-8- and IL-1beta-dependent shedding of the EGFR ligand in KATO III cells. METHODS: We established transfectants stably expressing alkaline phosphatase-tagged heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha, or amphiregulin precursors, and depleted ADAM proteins, using short interfering RNA against ADAM10, 12, or 17. We assessed shedding of EGFR ligands by measuring AP activities in the conditioned media after IL-1beta or IL-8 stimulation. EGFR activation was examined by immunoprecipitation and Western blotting using antiphosphotyrosine antibody. KB-R7785 and anti-IL-8 neutralizing antibody were used to inhibit activities of ADAMs and IL-8 action, respectively. RESULTS: IL-8 dose dependently released the EGFR ligands and transiently phosphorylated EGFR, with a peak at 15 minutes. KB-R7785 completely blocked IL-8-induced shedding and EGFR transactivation. Depletion of ADAM10 also dramatically reduced IL-8-induced shedding and EGFR transactivation, but depletion of ADAM12 and 17 did not. IL-1beta dose dependently enhanced shedding of HB-EGF, which was not blocked by KB-R7785 in the early phase. In the late phase, however, the EGFR transactivation was blocked by KB-R7785 and abrogated by anti-IL-8 neutralizing antibody. CONCLUSIONS: IL-8 induces shedding of EGFR ligands because of an ADAM10-dependent pathway in gastric cancer cells, whereas IL-1beta acts principally by an ADAM-independent pathway. IL-1beta-dependent prolonged EGFR transactivation involves multiple pathways, including an IL-8-dependent pathway.

ADAM Proteins↗

PubMed2004

Metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor and its pathophysiological roles.

Heparin-binding EGF-like growth factor (HB-EGF) exists as a membrane-anchored form (proHB-EGF) and as its soluble cleaved product (sHB-EGF). The conversion (ectodomain shedding) of proHB-EGF to sHB-EGF is tightly regulated by specific metalloproteinases. Ectodomain shedding plays a central role in GPCR-mediated EGFR transactivation. Antagonizing metalloproteinases can inhibit EGFR transactivation and might be of therapeutic value, for example in cardiac hypertrophy, skin remodeling and tumor growth.

Animals↗

PubMed2003

Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF.

Cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) via metalloprotease activation yields amino- and carboxy-terminal regions (HB-EGF and HB-EGF-C, respectively), with HB-EGF widely recognized as a key element of epidermal growth factor receptor transactivation in G protein-coupled receptor signaling. Here, we show a biological role of HB-EGF-C in cells. Subsequent to proteolytic cleavage of proHB-EGF, HB-EGF-C translocated from the plasma membrane into the nucleus. This translocation triggered nuclear export of the transcriptional repressor, promyelocytic leukemia zinc finger (PLZF), which we identify as an HB-EGF-C binding protein. Suppression of cyclin A and delayed entry of S-phase in cells expressing PLZF were reversed by the production of HB-EGF-C. These results indicate that released HB-EGF-C functions as an intracellular signal and coordinates cell cycle progression with HB-EGF.

Active Transport, Cell Nucleus↗

PubMed2003

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities.

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.

Animals↗

PubMed2003

Transactivation of epidermal growth factor receptor after heparin-binding epidermal growth factor-like growth factor shedding in the migration of prostate cancer cells promoted by bombesin.

BACKGROUND: A pathway consisting of bombesin, G-protein coupling receptors (GPCRs), metalloproteases, pro-heparin-binding epidermal growth factor (proHB-EGF), and epidermal growth factor receptor (EGFR) has been reported in prostate cancer cells. The occurrence of HB-EGF shedding from proHB-EGF in this pathway, however, has not been proven directly. In addition, it is still unclear how much this pathway contributes to the migration of prostate cancer cells. In this study, we tried to directly elucidate HB-EGF shedding in this pathway and to determine its contribution to the migration of prostate cancer cells. METHODS: RT-PCR and indirect immunofluorescence staining for HB-EGF and its receptors, such as EGFR and HER4/erbB4, were performed on PC-3 cells. The influences of bombesin, anti-EGFR neutralizing monoclonal antibody, HB-EGF, and HB-EGF shedding inhibitor on the migration of PC-3 cells were studied by means of in vitro wound assays. The amount of HB-EGF shed from PC-3 cells with alkaline phosphatase-tagged HB-EGF in the presence of bombesin was determined by measuring AP activity. Immunoprecipitations and phosphotyrosine Western blotting were performed to detect EGFR transactivated by bombesin. RESULTS: PC-3 expressed HB-EGF and EGFR, but not HER4/erbB4. PC-3 migrated in the presence of bombesin, but its migration was partly inhibited by the neutralizing antibody against EGFR. PC-3 also migrated in the presence of HB-EGF, but HB-EGF shedding inhibitor partly inhibited this phenomenon. HB-EGF was shed from PC-3 cells in the presence of bombesin, and this shedding was inhibited by HB-EGF shedding inhibitor. In addition, the EGFR on PC-3 was activated in the presence of bombesin and inactivated in the presence of HB-EGF shedding inhibitor. CONCLUSIONS: These results indicated that HB-EGF shedding and the following transactivation of EGFR occurs in this pathway and that this pathway partly contributes to the migration of prostate cancer cells.

Bombesin↗

PubMed2003

PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor.

A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.

ADAM Proteins↗

PubMed2003

Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function.

The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HB(del/del)) were generated to examine the role of HB-EGF in vivo. More than half of the HB(del/del) mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HB(del/del) mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF-ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HB(del/del) hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function.

Animals↗

PubMed2003

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy.

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. METHODS & RESULTS: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. CONCLUSIONS: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.

Animals↗

PubMed2003

Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor.

Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial-derived heparin binding EGF-like growth factor (HB-EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB-ligands demonstrated that primary ECs expressed only HB-EGF and neuregulin-1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB-EGF, but not neuregulin-1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB-EGF or inhibition of ErbB1 or ErbB2 blocked 70-90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin-1, an EC effector with a role in recruitment of SMC-like cells to vascular structures in vivo, enhanced EC-stimulated SMC migration by a mechanism involving up-regulation of endothelial HB-EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB-EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB-EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.

Angiogenesis Inducing Agents↗

PubMed2003

Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.

ADAM Proteins↗

PubMed2003

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta.

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.

Acetophenones↗