Extract of Oxytropis pseudoglandulosa inhibits vascular smooth muscle cell proliferation and migration via suppression of ERK1/2 and Akt signaling pathways1.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration accelerate the development of occlusive vascular disease. Therefore, finding a means to control the aberrant proliferation and migration of VSMCs has own clinical significance. In the present study, we examined the feasibility of using extract from medicinal plant Oxytropis pseudoglandulosa (OG) to control pathologic proliferation and migration of VSMCs, which never have been tested. Our data indicate that the extract of OG significantly suppressed proliferation and migration of VSMCs without cytotoxic effect, suggesting the OG extract may be an alternative agent to effectively control the aberrant VSMC proliferation and migration without any serious adverse effect. These data suggest that the extract of OG may be a potent therapeutic agent for the treatment of occlusive vascular disease and warrant further studies to identify the major acting ingredient and to validate in vivo efficacy.

7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d] pyrimidin-4-ylamine inhibits the proliferation and migration of vascular smooth muscle cells by suppressing ERK and Akt pathways.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury significantly contributes to the development of occlusive vascular disease. Therefore, inhibiting the proliferation and migration of VSMCs is a validated therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. In the present study, we screened chemical compounds for their anti-proliferative effects on VSMCs using multiple approaches, such as MTT assays, wound healing assays, and trans-well migration assays. Our data indicate that 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d] pyrimidin-4-ylamine, a lymphocyte-specific protein tyrosine kinase (Lck) inhibitor, significantly inhibited both VSMC proliferation and migration. 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine suppresses VSMC proliferation and migration via down-regulating the protein kinase B (Akt) and extracellular signal regulated kinase (ERK) pathways, and it significantly decreased the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 and, the phosphorylation of retinoblastoma protein (pRb). Additionally, 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d] pyrimidin-4-ylamine suppressed the migration of VSMCs from endothelium-removed aortic rings, as well as neointima formation following rat carotid balloon injury. The present study identified 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its more detailed molecular mechanisms, such as its primary target, and to further validate its in vivo efficacy as a therapeutic agent for pathologic vascular conditions, such as restenosis and atherosclerosis.

A spleen tyrosine kinase inhibitor attenuates the proliferation and migration of vascular smooth muscle cells.

BACKGROUND: Pathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay. RESULTS: Among 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings. CONCLUSION: The present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis.

A spleen tyrosine kinase inhibitor attenuates the proliferation and migration of vascular smooth muscle cells

BACKGROUND: Pathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay. RESULTS: Among 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings. CONCLUSION: The present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis.

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway.

AIM OF THE STUDY: Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa. MATERIALS AND METHODS: Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used. RESULTS: CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants. CONCLUSIONS: These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.

Therapeutic effect of magnesium lithospermate B on neointimal formation after balloon-induced vascular injury.

Vascular smooth muscle cell (VSMC) proliferation and migration in response to platelet-derived growth factor (PDGF) play an important role in the development of atherosclerosis and restenosis. Recent evidence indicates that PDGF increases intracellular levels of reactive oxygen species in VSMCs and that both PDGF-induced VSMC proliferation and migration are reactive oxygen species-dependent. Danshen is a representative oriental medicine used for the treatment of vascular disease. Previously, we reported that magnesium lithospermate B, an active component of Danshen, is a potent antioxidant. Thus we investigated the therapeutic potential of magnesium lithospermate B in neointimal formation after carotid artery injury in rats along with its effects on the PDGF signaling pathway for stimulating VSMC proliferation and migration in vitro. PDGF is dimeric glycoprotein composed of two A or two B chains. In this study, we used PDGF-BB, which is one of the isoforms of PDGF (i.e., PDGF-AA, PDGF-BB, and PDGF-AB). Our results demonstrated that magnesium lithospermate B directly scavenged reactive oxygen species in a xanthine/xanthine oxidase system and reduced PDGF-BB-induced intracellular reactive oxygen species generation in VSMCs. In a rat carotid artery balloon injury model, magnesium lithospermate B treatment (10 mg/kg/day, i.p) showed a significant effect on the prevention of neointimal formation compared with vehicle treatment. In cultured VSMCs, magnesium lithospermate B significantly attenuated PDGF-BB-induced cell proliferation and migration as measured by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and transwell migration assays, respectively. Further, magnesium lithospermate B inhibited PDGF-BB-induced phosphorylation of phospatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways by scavenging reactive oxygen species. Together, these data indicated that magnesium lithospermate B, a potent reactive oxygen species scavenger, prevented both injury-induced neointimal formation in vivo and PDGF-BB-induced VSMC proliferation and migration in vitro, suggesting that magnesium lithospermate B may be a promising agent to prevent atherosclerosis and restenosis following angioplasty.

Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage.

The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IkappaB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-kappaB p65 subunits, which correlated with PLBF's inhibitory effects on IkappaBalpha phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-kappaB (NF-kappaB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene expression via ROS-based NF-kappaB and MAPKs activation. Thus, PLBF may provide a potential therapeutic approach for inflammation-associated disorders.

Ethanol extract of Inonotus obliquus inhibits lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells.

Inonotus obliquus (Pers.:Fr.) Pil. is a white rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes. Extracts and fractions of this fungus have been known to have biological activities, including antimutagenic, anticancer, antioxidative, and immunostimulating effects. Recently, there have been reports that the anti-inflammatory and antinociceptive properties of the methanol extract of I. obliquus may be due to the inhibition of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) expression via the down-regulation of nuclear factor kappaB (NF-kappaB) binding activity. However, the effects of I. obliquus on Akt and mitogen-activated protein kinase (MAPK) activation of inflammatory mediator production have not yet been elucidated. In the present study, a 70% ethanol extract of I. obliquus (IOE70) showed antioxidative effects. We also tested the ability of the I. obliquus extract to inhibit the inflammatory cascades in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. The NO inhibition of IOE70 was better than that of other ethanol extracts from I. obliquus. To investigate the mechanism by which IOE 70 inhibits NO production and iNOS and COX-2 expression, we examined the activations of IkappaBalpha, Akt, and c-Jun NH(2) -terminal kinase (JNK) in LPS-activated macrophages. IOE70 markedly inhibited the phosphorylation of IkappaBalpha, Akt, and MAPKs in dose-dependent manners in LPS-activated macrophages. Taken together, these experiments demonstrated that IOE70 inhibition of LPS-induced expression of iNOS and COX-2 protein is mediated by Akt and JNK. Based on our findings, the most likely mechanism that can account for this biological effect of IOE70 involves the inhibition of NF-kappaB through the phosphatidylinositol 3-kinase/Akt/IkappaB pathway and the inhibition of JNK activation. Thus, IOE70 might have useful clinical applications in the management of inflammatory diseases and may also be useful as a medicinal food.

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.