The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogenactivated protein kinase kinase 1/2, prevented the VPAinduced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvationinduced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.
Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-{methyl[1-(2-naphthoyl)piperidin-4-yl]amino}carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC50 of ∼600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca2+ mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.
We have recently shown that in polymorphonuclear leukocytes, 11‐keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen‐activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions.
We found that β‐BA (10 μM), the 11‐methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal‐regulated kinase (ERK)2, and Akt. These effects of β‐BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen.
Based on inhibitor studies, β‐BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol‐1,4,5‐trisphosphate pathway, and involves Src family kinase signalling.
Investigation of platelet functions revealed that β‐BA (10 μM) strongly stimulates the platelet‐induced generation of thrombin in an ex‐vivo in‐vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+‐dependent manner.
In contrast to β‐BA, the 11‐keto‐BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin.
In summary, β‐BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation.
British Journal of Pharmacology (2005) 146, 514–524. doi:
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