Lynda A. Sellers

Modulating Angiogenesis The Yin and the Yang in Ginseng

Background—Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. Methods and Results—A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct “sterol ginsenoside” fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase→Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. Conclusions—The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing.

Immunohistochemical localization of the somatostatin sst2(a) receptor in the rat brain and spinal cord

The neuropeptide somatostatin is widely distributed in the CNS and is believed to play a role as a neurotransmitter or a neuromodulator. Somatostatin mediates its actions by the binding of the peptide to high affinity membrane receptors. The genes for five somatostatin receptor types have been cloned recently and Northern blotting and in situ hybridization studies have shown that the transcripts of all five types are expressed in the CNS. Here we report the cellular distribution of somatostatin sst2(a) receptor protein in the adult rat CNS, using a polyclonal anti-peptide antibody directed against a portion of the C-terminal domain of the receptor. The specificity of the affinity-purified antibody was demonstrated by Western blotting and immunolabelling of cells transfected with a hemagglutinin epitope-tagged version of the sst2(a) receptor. Immunohistochemistry showed a distinct distribution of the receptor protein in the rat brain. Cells and processes were labelled in a number of areas, including the basolateral amygdala, the locus coeruleus, the endopiriform nucleus, the deep layers of the cerebral cortex, the subiculum, the claustrum, the habenula, the interpenduncular nucleus, the hippocampus and the central grey. In the spinal cord, the substantia gelatinosa showed strongly-labelled cell bodies and their processes. This study provides an improved understanding of the distribution of the sst2(a) receptor in rat brain.

Increased MAP kinase activity in Alzheimer's and Down syndrome but not in schizophrenia human brain

Abnormal phosphorylation of tau is a feature of Alzheimers disease (AD), which develops prematurely in Down syndrome (DS) patients. Cognitive impairment is also recognized as a clinical characteristic of schizophrenia, which does not appear to be associated with tau‐aggregate formation. Several kinases can phosphorylate tau in cell‐free assays. Here we show increased activity of mitogen‐activated protein kinases (MAPKs) (including ERK1/2, SAPKs and p38) in post mortem AD and DS brains, which could not be accounted for by expression changes. In contrast, glycogen synthase kinase‐3 activity (GSK‐3αβ) was reduced significantly. Examination of tau in AD and DS using antibodies selective for MAPK phosphorylation sites showed increased immunoreactivity. In addition, phosphorylation of S199, reportedly a selective substrate for cyclin‐dependent kinase‐5 (cdk5) or GSK‐3αβ was only observed in AD samples, which showed a concomitant increase in the expression of p25, the enhancing cofactor for cdk5 activity. However, in schizophrenia brain, MAPK‐phosphorylated tau was unchanged compared to matched controls, despite similar expression levels to those in AD. The activities of the MAPKs and GSK‐3αβ were also unchanged. These data demonstrate that in AD and DS, enhanced MAPK activity, which has an established role in regulating neuronal plasticity and survival, can account for irregular tau phosphorylation, and that the molecular processes involved in these neurodegenerative disorders are distinct from those in schizophrenia. These data also question the significance of GSK‐3αβ, as much previous work carried out in vitro has placed this kinase as a favoured candidate for involvement in the pathological phosphorylation of tau.

Hepatocyte Growth Factor/Scatter Factor Can Induce Angiogenesis Independently of Vascular Endothelial Growth Factor

Objective—Hepatocyte growth factor/scatter factor (HGF/SF) promotes vascular endothelial growth factor (VEGF) expression and induces angiogenesis in multiple pathological conditions. The present study was designed to delineate the HGF/SF and VEGF signaling cascades during angiogenesis by using PTK787, a selective VEGF receptor antagonist. Methods and Results—PTK787 produced a concentration-dependent (10−8 to 10−6 mol/L) inhibition of VEGF-induced angiogenesis, without altering the basal or HGF/SF-induced response in vitro. In contrast, the nonspecific kinase inhibitor genistein blocked the HGF/SF-induced effect. Both VEGF and HGF/SF induced a rapid phosphorylation of extracellular receptor kinases-1 and -2 (ERKs) and Akt. PTK787 inhibited the VEGF-induced activation of Akt and ERKs, without affecting the HGF/SF-induced phosphorylation. Treatment with VEGF and HGF/SF increased total neovascularization in a murine scaffold granuloma model, but no additive or synergistic interactions were observed. PTK787 (50 mg/kg) blocked the VEGF-induced response without altering the basal or HGF/SF-induced neovascularization. Conclusions—We demonstrate that HGF/SF can induce angiogenesis independently of VEGF, possibly through the direct activation of the Akt and ERKs. These results demonstrate the necessity of a multitargeted approach for the rational design of newer therapies to inhibit pathophysiological angiogenesis.

Prostacyclin Induces Apoptosis of Vascular Smooth Muscle Cells by a cAMP-Mediated Inhibition of Extracellular Signal-Regulated Kinase Activity and Can Counteract the Mitogenic Activity of Endothelin-1 or Basic Fibroblast Growth Factor

Abstract— Prostanoids can suppress vascular smooth muscle cell (VSMC) proliferation, but the mechanism through which this is mediated has not been identified. In this study, we show rat aortic VSMCs to express the EP1, EP2, EP3, EP4, and IP receptors. The EP4 receptor–specific agonist, 11-deoxy-PGE1, induced a time-dependent phosphorylation of protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 in serum-depleted (0.1%) VSMCs, whereas the EP2 receptor agonist, butaprost, was without effect. PGI2 or iloprost at the IP receptor inhibited basal ERK phosphorylation with IC50 values of ≈10 nmol/L. Iloprost also attenuated the sustained activation of ERK induced by endothelin-1 or basic fibroblast growth factor (bFGF). Endothelin-1 or bFGF significantly increased the number of VSMCs counted 24 hours later compared with basal, and both responses were blocked by the MEK inhibitor, U0126, or iloprost. Under basal conditions, U0126 or iloprost reduced the number of viable cells and increased caspase-3 activity, which could be reversed by coapplication with endothelin-1, bFGF, or the adenylate cyclase inhibitor, SQ22536. Endothelin-1, bFGF, or SQ22536 prevented the depression to below basal levels of ERK phosphorylation induced by iloprost. Forskolin activated caspase-3 and attenuated basal ERK phosphorylation, which were prevented by SQ22536, endothelin-1, or bFGF. These data suggest that iloprost induces apoptosis via a cAMP-mediated suppression of ERK activity. In turn, this apoptotic response can be blocked by a mitogenic stimulus that re-establishes ERK activity back to basal levels, but at the expense of any concomitant proliferative activity. However, ERK stimulation by a selective EP4 receptor agonist, suggests that prostanoids may have diverse and complex roles in VSMC physiology.

Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms

Thymidine phosphorylase (TP) is elevated in the plasma of cancer patients, and has been implicated in pathophysiological angiogenesis. However, the downstream signals underlying this implication remain obscure. The purpose of the present study was to examine the effects of TP on the neovascularisation response in vitro and in vivo. Both TP and its catalytic product, 2‐deoxy‐D‐ribose‐1‐phosphate, and downstream 2‐deoxy‐D‐ribose (2‐DDR) promoted endothelial tubulogenesis in vitro, and the regeneration of a wounded monolayer of endothelial cells without exerting any mitogenic effect. In vivo, both TP and 2‐DDR promoted the development of functional vasculature into an avascular sponge. A TP inhibitor, 6‐amino‐5‐chlorouracil, was able to partially reverse the effects of TP, but had no effect on the 2‐DDR‐induced angiogenesis. Enhanced monolayer regeneration was observed with TP‐cDNA‐transfected bladder carcinoma cells. The transfection of TP‐cDNA, however, did not confer any proliferative advantage. The regeneration of TP overexpressing cells was associated with a time‐dependent expression of the enzyme haeme‐oxygenase (HO‐1). The present study demonstrates that both TP and its ribose‐sugar metabolites induce angiogenesis by mediating a cohesive interplay between carcinoma and endothelial cells. The induction of HO‐1 in TP‐transfected cells suggests that it could be a possible downstream signal for the angiogenic effects of TP. Furthermore, reducing sugars have been shown to induce oxidative stress, and ribose could be a possible cause for the upregulation of HO‐1, which has been implicated in the release of angiogenic factors. Therefore, we postulate that 2‐DDR could be mediating the angiogenic effects of TP possibly through an oxidative stress mechanism and additionally getting integrated in the endothelial metabolic machinery.

Activated G Protein-coupled Receptor Induces Tyrosine Phosphorylation of STAT3 and Agonist-selective Serine Phosphorylation via Sustained Stimulation of Mitogen-activated Protein Kinase RESULTANT EFFECTS ON CELL PROLIFERATION

The peptide hormone somatostatin exhibits antiproliferative activity by interacting with the G protein-coupled sst2 or sst5 receptor types. We show here that somatostatin at the human recombinant sst4 receptor induced a concentration-dependent increase in proliferation (EC50 20 nm) with a maximal response 5-fold greater than that produced by its synthetic analog, L-362,855. Analysis of the phosphorylation status of extracellular signal-regulated kinase (ERK)1 and ERK2 showed temporal differences in the changes evoked by the agonists. Phosphorylation induced by somatostatin (100 nm) peaked 10 min after the application and produced a response that continued for at least 4 h. In contrast, L-362,855 (1 μm) showed transient phosphorylation that had declined to basal levels by 1 h. However, both agonists induced rapid and sustained tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) which was pertussis toxin-insensitive. Serine phosphorylation of STAT3 was only apparent after somatostatin treatment and was abolished by pertussis toxin or PD 98059, together with the associated increases in proliferation. Mitogen-activated protein/ERK kinase-1 inhibition also decreased the time interval over which somatostatin-induced ERK phosphorylation was observed (<2 h). We conclude that the difference in the magnitude of the proliferative response evoked by the two agonists at the sst4 receptor can be accounted for by their differential ability to phosphorylate STAT3 on serine residues and supports the concept that selective signaling can be achieved through pharmacological diversity.

Signalling pathways influencing basal and H2O2-induced P-glycoprotein expression in endothelial cells derived from the blood–brain barrier

The drug transporter, P‐glycoprotein (P‐gp) on brain microvessel endothelium, influences movement of lipophilic substances in and out of the brain. Pathways regulating P‐gp expression, both basal and hydrogen peroxide (H2O2)‐induced, are here examined in primary cultured rat brain endothelial cells. Activation of extracellular‐signal regulated kinases (ERK1/2), protein kinase C (PKC), the p46 isoform of stress‐activated protein kinase (SAPK) and its downstream transcription factor, c‐Jun, occurred in a time‐ and concentration‐dependent manner following exposure of cells to H2O2 with concomitant increases in P‐gp expression. Blockade of ERK activation with U0126, of PKC with Gö6976 and of SAPK with SP600125 decreased basal P‐gp but did not abolish the H2O2‐induced increase. Blockade of Akt with PI3‐kinase inhibitor, LY294002, lowered basal P‐gp and prevented the H2O2‐induced increase. Inhibition of nuclear factor‐κB (NF‐κB), either by blocking dissociation from its inhibitory factor, IκB, with MG132 or its nuclear translocation with SN50 enhanced basal P‐gp, obscuring the H2O2‐induced increase. H2O2 itself produced no detectable activation of IκB, but inhibited that induced by 5 ng/mL tumour necrosis factor‐α (TNF‐α). P‐gp expression may involve positive inputs from ERK1/2, SAPK, Akt and PKC and inhibitory influences of NF‐κB. By depressing NF‐κB signalling, H2O2 may still augment P‐gp expression when ERK1/2, PKC or SAPK are inhibited.

Receptor Isoforms Mediate Opposing Proliferative Effects through Gβγ-Activated p38 or Akt Pathways

ABSTRACT The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst2 receptor isoforms, which couple to identical Gα subunit pools (Gαi3 > Gαi2 >> Gα0), were both inhibited following βγ sequestration. The sst2(a) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70 rsk induced by sst2(b) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst2(a) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21 cip1 was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst2(a) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst2(a) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst2 receptor isoforms may determine the selection of appropriate βγ-pairings necessary for interaction with distinct kinase cascades.

Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant

1 The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy‐termini only. The murine sst2(b) receptor was reported to be less prone to agonist‐induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full‐length protein in CHO‐K1 cells for functional characterization. 2 This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy‐terminus differing in composition to the 38 amino acid C‐terminus of the rat sst2(a) receptor. 3 In CHO‐K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration‐dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre‐treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration‐effect curves with pEC50 values of 8.3 (sst2(a)) and 8.4 (sst2(b)). 4 SRIF (3 pM–3 nM) also caused concentration‐dependent inhibition of forskolin‐stimulated cyclic AMP formation in CHO‐sst2(a) cells (pIC50 10.5) and CHO‐sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell‐shaped concentration‐effect curves. Following pre‐treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5 Both the SRIF‐induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist‐induced desensitization, but this was less apparent following pre‐treatment with Ptx. 6 This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx‐sensitive as well as ‐insensitive G proteins and are equally prone to agonist‐induced desensitization.