Susan C. Olson

Resveratrol Prevents Monocrotaline-induced Pulmonary Hypertension in Rats

Proliferation of pulmonary arterial smooth muscle cells, endothelial dysfunction, oxidative stress, and inflammation promotes the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exerts antioxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteries remain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonary hypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment (25 mg/kg per day, PO, from day 1 postmonocrotaline) attenuated right ventricular systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (tumor necrosis factor-α, interleukin 1β, interleukin 6, and platelet-derived growth factor-α/β), and limited leukocyte infiltration in the lung. Resveratrol also inhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol increased expression of endothelial NO synthase, decreased oxidative stress, and improved endothelial function in small pulmonary arteries. Pulmonary hypertension was associated with an upregulation of NAD(P)H oxidase in small pulmonary arteries, which was significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory, antioxidant, and antiproliferative effects in the pulmonary arteries, which may contribute to the prevention of pulmonary hypertension.

Regulation of G1/S transition and induction of apoptosis in HL‐60 leukemia cells by fenretinide (4HPR)

We previously reported that all‐trans retinoic acid (RA) and fenretinide (4HPR) suppress HL‐60 leukemia cell growth and cause partial cell arrest in the G1‐to‐S phase. Moreover, 4HPR but not RA induces apoptosis in HL‐60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40–75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR‐treated cells. Evidence for 4HPR‐induced apoptosis comes from (1) cleavage of the enzyme poly(ADP‐ribose) polymerase (PARP) to an 89‐kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3′‐ends of DNA. Overnight pretreatment with 0.5–5.0 μM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid‐derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2‐fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR‐treated HL‐60 cells, the effects of fumonisin B1, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25–100 μM fumonisin B1 resulted in a dose‐dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR‐induced increase in ceramide. Int. J. Cancer 78:53–61, 1998.© 1998 Wiley‐Liss, Inc.

Induced cytoskeletal changes in bovine pulmonary artery endothelial cells by resveratrol and the accompanying modified responses to arterial shear stress

BackgroundAtherosclerosis and coronary heart disease (CHD) are significant contributors to morbidity and mortality in developed countries. A noted exception is the low mortality of CHD in France, particularly the southwest region. This phenomenon, commonly referred to as the French paradox, may be associated with high consumption of red wine. We investigate whether the cardioprotective activity of red wine may involve the grape skin-derived polyphenol, resveratrol. We further test the possibility that resveratrol acts by modulating structural and functional changes in endothelial cells lining the blood vessel wall.ResultsBovine pulmonary artery endothelial cells (BPAEC) were incubated with resveratrol, with and without concurrent exposure to simulated arterial shear stress. Resveratrol significantly affected proliferation and shape of BPAEC; growth was suppressed and cells became elongated, based on morphologic analysis of rhodamine-conjugated phalloidin stained F-actin by confocal microscopy. Using selective signaling inhibitors, we showed that the resveratrol-induced cellular phenotype was dependent on intracellular calcium and tyrosine kinase activities, and assembly of actin microfilaments and microtubules, but was unrelated to PKC activity. Exposure to simulated arterial flow revealed that, whereas controls cells easily detached from the culture support in a time-dependent manner, resulting in total cell loss after a 5 min challenge with simulated arterial flow conditions, a significant percentage of the treated cells remained attached to the cultured plastic coverslips under identical experimental conditions, suggesting that they adhered more strongly to the surface. Western blot analysis shows that whereas cells treated with 25 μM and 100 μM resveratrol had no change in total ERK1/2, treatment did result in an increase in phosphorylated ERK1/2, which probably involved stabilization of the active enzyme. An increase in nitric oxide synthase expression was detected as early as 6 h and persisted for up to 4 days of treatment.ConclusionsResults of our studies show that resveratrol interacts with endothelial cells in vitro to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

Peroxide generation by p47phox-Src activation of Nox2 has a key role in protein kinase C-induced arterial smooth muscle contraction

Protein kinase C (PKC) stimulation of NAD(P)H oxidases (Nox) is an important component of multiple vascular disease processes; however, the relationship between oxidase activation and the regulation of vascular smooth muscle contraction by PKC remains poorly understood. Therefore, we examined the signaling cascade of PKC-elicited Nox activation and the role of superoxide and hydrogen peroxide in mediating PKC-induced vascular contraction. Endothelium-denuded bovine coronary arteries showed a PKC-dependent basal production of lucigenin (5 muM)-detected Nox oxidase-derived superoxide, which was stimulated fourfold by PKC activation with 10 muM phorbol 12,13-dibutyrate (PDBu). PDBu appeared to increase superoxide generation by Nox2 through both p47(phox) and peroxide-dependent Src activation mechanisms based on the actions of inhibitors, properties of Src phosphorylation, and the loss of responses in aorta from mice deficient in Nox2 and p47(phox). The actions of inhibitors of contractile regulating mechanisms, scavengers of superoxide and peroxide, and responses in knockout mouse aortas suggest that a major component of the contraction elicited by PDBu appeared to be mediated through peroxide derived from Nox2 activation stimulating force generation through Rho kinase and calmodulin kinase-II mechanisms. Superoxide generated by PDBu also attenuated relaxation to nitroglycerin. Peroxide-derived from Nox2 activation by PKC appeared to be a major contributor to the thromboxane A(2) receptor agonist U46619 (100 nM)-elicited contraction of coronary arteries. Thus a p47(phox) and Src kinase activation of peroxide production by Nox2 appears to be an important contributor to vascular contractile mechanisms mediated through activation of PKC.

Biochemistry and cell biology of phospholipase D in human neutrophils

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.

Smurf1 ubiquitin ligase causes downregulation of BMP receptors and is induced in monocrotaline and hypoxia models of pulmonary arterial hypertension

Reduced bone morphogenetic protein (BMP) receptor (BMPR) expression and BMP signaling have been implicated in vascular cell proliferation and remodeling associated with pulmonary arterial hypertension (PAH). The low penetrance of the BMPR II disease gene in familial PAH suggests that additional genetic or environmental factors are involved in clinical manifestation of PAH. Smurf1 ubiquitin ligase, together with inhibitory SMAD 6/7, forms a negative feedback loop for the attenuation of BMP signals by downregulating BMPR and signaling molecules and, in addition, functions in the integration of MAPK/Ras mitogenic pathways. The present study found that Smurf1 was significantly elevated in pulmonary arteries of monocrotaline and hypoxia-induced PAH rats. In the pulmonary artery of hypoxia-exposed mice, elevation of Smurf1 and SMAD7 was correlated with reduced expression of BMPR II protein. Over-expression of Smurf1 in cultured cells induced ubiquitination and degradation of BMPR I and II whereas ligase-inactive Smurf1 reduced ubiquitination and elevated their protein levels, thus serving a dominant-negative function. Smurf1-induced receptor degradation was inhibited by both proteasomal and lysosomal inhibitors. Thus, Smurf1 reduces steady-state levels of BMPRs by ubiquitination and subsequent degradation involving proteasomes and lysosomes. Therefore, these results show that Smurf1 induction could be a key event for triggering downregulation of BMP signaling and causing vascular cell proliferation and remodeling in PAH and that abrogating Smurf1 function could be a strategy for PAH therapeutics.

Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model

BACKGROUND C-fos and c-jun are 2 immediate early genes that have been implicated in the stimulation of vascular smooth muscle cell proliferation and migration. In previous experiments in our laboratory with a rat vein graft model a 2- to 3-fold increase of messenger RNA of c-fos and c-jun were noted 1 hour after vein graft perfusion. Because c-fos and c-jun are up-regulated after the perfusion of vein grafts, the purpose of this study was to delineate the temporal expression of c-fos and c-jun protein and to study the effect of antisense oligonucleotides (ASO) to c-fos and c-jun on intimal thickening observed in this model. METHODS Sprague-Dawley rats underwent bilateral interposition femoral artery grafts with use of the superficial epigastric vein, which was harvested from 15 minutes up to 2 weeks and analyzed by Western blot for Fos and Jun protein. Additional rats underwent bypasses and at the time of the procedure 1 graft was treated with a pluronic gel containing an ASO to c-fos, c-jun, or sense and the contralateral side was treated with pluronic gel only. The vein grafts were harvested 2 weeks after the procedure and perfusion fixed. After longitudinal sectioning, the intimal and total wall thicknesses were measured in the perianastamotic and midgraft regions by a morphometric digitizing microscope and the statistics were analyzed by a paired Students t test. RESULTS Protein analysis by Western blot showed that c-fos levels rose quickly within 2 hours and leveled at 6 hours 40-fold above basal levels after vein graft perfusion. Similarly, c-jun levels rose 10-fold above basal levels after 15 minutes and peaked at 2 hours 120-fold above basal levels. The treatment of the vein grafts with these ASOs resulted in a reduction of about 30% in the thickness of the intimal layer and the total wall thickness in both the perianastomotic and the midgraft regions, which was statistically significant different from control veins. CONCLUSION These results indicate a possible therapeutic role for ASO to immediate early genes in the treatment of vein graft intimal hyperplasia.

Angiotensin type 1 receptor is linked to inhibition of nitric oxide production in pulmonary endothelial cells

We previously demonstrated that angiotensin II (Ang II) stimulates an increase in nitric oxide synthase (NOS) mRNA levels, eNOS protein expression and NO production via the type 2 (AT2) receptor, whereas signaling via the type 1 (AT1) receptor negatively regulates NO production in bovine pulmonary artery endothelial cells (BPAECs). In the present study, we investigated the components of the AT1 receptor-linked signaling pathway(s) that are involved in the downregulation of eNOS protein expression in BPAECs. Treatment of BPAECs with either AT1 receptor antagonists or an anti-AT1 receptor antibody induced eNOS protein expression. Furthermore, intracellular delivery of GP-Antagonist-2A, an inhibitor of Galphaq proteins, and treatment of BPAECs with U73122, a phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, enhanced eNOS protein expression. Treatment of BPAECs with the cell-permeable calcium chelator, BAPTA/AM, increased eNOS protein expression at 8 h, while increasing intracellular calcium with either thapsigargin or A23187 prevented Ang II-induced eNOS protein expression. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, completely prevented Ang II-stimulated eNOS protein expression at 8 h, whereas depletion of PKC by long-term treatment with PMA, induced eNOS protein expression. Treatment of BPAECs with a PKCalpha-specific inhibitor or transfection of BPAECs with an anti-PKCalpha neutralizing antibody stimulated eNOS protein expression. Conversely, rottlerin, a PKCdelta specific isoform inhibitor had no effect on basal or Ang II-stimulated eNOS protein expression. Moreover, treatment of BPAECs with U73122, BAPTA/AM and PKCalpha-specific inhibitors increased NO production at 8 h. In conclusion, Ang II downregulates eNOS protein expression via an AT1 receptor-linked pathway involving Galphaq/PLC/calcium/PKCalpha signaling pathway in BPAECs.

Resveratrol and its metabolites modulate cytokine‐mediated induction of eotaxin‐1 in human pulmonary artery endothelial cells

Coronary heart disease (CHD) is a leading cause of death in many developed countries. Evidence has long implicated endothelial injury and inflammation as apical events in the pathogenesis of atherosclerosis, the primary cause of CHD. Numerous risk factors contribute to a damaged, inflamed endothelium. Conversely, cardioprotective agents targeting the dysfunctional endothelium have also been identified, notably from dietary sources. We have used cultured human pulmonary artery endothelial cells (HPAECs) to test the diet‐mediated cardioprotective hypothesis. In this review, we summarize our recent findings on control of transcription and expression of inflammation biomarker eotaxin‐1 in HPAECs exposed to single or combined proinflammatory cytokines interleukin‐13 (IL‐13) and tumor necrosis factor–α (TNF‐α), and attenuation of the observed eotaxin‐1 responses by prior or simultaneous treatment with resveratrol and its metabolites. Control of eotaxin‐1 gene regulation may be considered an in vitro model to evaluate agents linking cardioprotection with endothelial cell damage and inflammation.