Prostacyclin Generation Research Articles

Pathogenesis of cardiovascular diseases: effects of mitochondrial CF6 on endothelial cell function.

Cardiovascular disease (CVD) stands as a predominant global cause of morbidity and mortality, necessitating effective and cost-efficient therapies for cardiovascular risk reduction. Mitochondrial coupling factor 6 (CF6), identified as a novel proatherogenic peptide, emerges as a significant risk factor in endothelial dysfunction development, correlating with CVD severity. CF6 expression can be heightened by CVD risk factors like mechanical force, hypoxia, or high glucose stimuli through the NF-κB pathway. Many studies have explored the CF6-CVD relationship, revealing elevated plasma CF6 levels in essential hypertension, atherosclerotic cardiovascular disease (ASCVD), stroke, and preeclampsia patients. CF6 acts as a vasoactive and proatherogenic peptide in CVD, inducing intracellular acidosis in vascular endothelial cells, inhibiting nitric oxide (NO) and prostacyclin generation, increasing blood pressure, and producing proatherogenic molecules, significantly contributing to CVD development. CF6 induces an imbalance in endothelium-dependent factors, including NO, prostacyclin, and asymmetric dimethylarginine (ADMA), promoting vasoconstriction, vascular remodeling, thrombosis, and insulin resistance, possibly via C-src Ca2+ and PRMT-1/DDAH-2-ADMA-NO pathways. This review offers a comprehensive exploration of CF6 in the context of CVD, providing mechanistic insights into its role in processes impacting CVD, with a focus on CF6 functions, intracellular signaling, and regulatory mechanisms in vascular endothelial cells.

Widening the Prostacyclin Paradigm: Tissue Fibroblasts Are a Critical Site of Production and Antithrombotic Protection.

Prostacyclin is a fundamental signaling pathway traditionally associated with the cardiovascular system and protection against thrombosis but which also has regulatory functions in fibrosis, proliferation, and immunity. Prevailing dogma states that prostacyclin is principally derived from vascular endothelium, although it is known that other cells can also synthesize it. However, the role of nonendothelial sources in prostacyclin production has not been systematically evaluated resulting in an underappreciation of their importance relative to better characterized endothelial sources. To address this, we have used novel endothelial cell-specific and fibroblast-specific COX (cyclo-oxygenase) and prostacyclin synthase knockout mice and cells freshly isolated from mouse and human lung tissue. We have assessed prostacyclin release by immunoassay and thrombosis in vivo using an FeCl3-induced carotid artery injury model. We found that in arteries, endothelial cells are the main source of prostacyclin but that in the lung, and other tissues, prostacyclin production occurs largely independently of endothelial and vascular smooth muscle cells. Instead, in mouse and human lung, prostacyclin production was strongly associated with fibroblasts. By comparison, microvascular endothelial cells from the lung showed weak prostacyclin synthetic capacity compared with those isolated from large arteries. Prostacyclin derived from fibroblasts and other nonendothelial sources was seen to contribute to antithrombotic protection. These observations define a new paradigm in prostacyclin biology in which fibroblast/nonendothelial-derived prostacyclin works in parallel with endothelium-derived prostanoids to control thrombotic risk and potentially a broad range of other biology. Although generation of prostacyclin by fibroblasts has been shown previously, the scale and systemic activity was unappreciated. As such, this represents a basic change in our understanding and may provide new insight into how diseases of the lung result in cardiovascular risk.

Vascular lipid droplets formed in response to TNF, hypoxia, or OA: biochemical composition and prostacyclin generation

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI2) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI2 generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI2 generation. In conclusion, vascular LDs formation and ATGL-dependent PGI2 generation represent a universal response to vascular proinflammatory insult.

Abstract 14698: Identifying Sources of Prostacyclin Outside the Vascular Wall and Their Contribution to Cardiovascular Protection

Introduction: Prostacyclin is a key anti-thrombotic hormone synthesised through cyclooxygenase (COX) enzymes. Historically, its production has been almost universally associated with the endothelium however the role of non-vascular sources has not been systematically evaluated due to a lack of suitable tools. This has resulted in an underappreciation of its cardiovascular biology and role in other contexts. Hypothesis: We hypothesised that cells outside the vascular wall contribute to generation of prostacyclin which can enter the circulation to provide anti-thrombotic protection. Methods: Global, endothelial and fibroblast COX1 or COX2 knockout mice were studied alongside freshly isolated healthy human lung tissue and primary cell cultures. Prostacyclin levels in plasma and released from cells/tissue ex vivo were measured by ELISA. Individual cell populations were isolated by FACS. Thrombosis in vivo was measured using a FeCl 3 -carotid artery injury model. Results: Vascular endothelial COX1 deletion strongly reduced prostacyclin release from mouse aorta and heart, but not from isolated lung, colon, kidney or spleen (Fig A). This non-vascular prostacyclin required COX1 as it was abolished by global COX1 and not COX2 deletion. Freshly isolated lung fibroblasts exhibited greater prostacyclin synthesis than endothelial or other cell types (Fig B) and this was confirmed in primary human lung cell cultures. Deletion of COX1 from fibroblasts in mice reduced lung prostacyclin release (Fig C) and plasma prostacyclin levels (Fig D) and this was associated with accelerated thrombotic occlusion after carotid artery injury (Fig E). Conclusions: These findings define a new paradigm in prostacyclin biology in which fibroblast/non-vascular-derived prostacyclin works in parallel with endothelium-derived prostanoids to control thrombosis. This may have particular relevance to understanding how local diseases of the lung result in cardiovascular risk.

Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation

Endothelial cyclooxygenase-1-derived prostanoids, including prostacyclin, have clear cardioprotective roles associated with their anti-thrombotic potential but have also been suggested to have paradoxical pathological activities within arteries. To date it has not been possible to test the importance of this because no models have been available that separate vascular cyclooxygenase-1 products from those generated elsewhere. Here, we have used unique endothelial-specific cyclooxygenase-1 knockout mice to show that endothelial cyclooxygenase-1 produces both protective and pathological products. Functionally, however, the overall effect of these was to drive pathological responses in the context of both vasoconstriction in vitro and the development of atherosclerosis and vascular inflammation in vivo. These data provide the first demonstration of a pathological role for the vascular cyclooxygenase-1 pathway, highlighting its potential as a therapeutic target. They also emphasize that, across biology, the role of prostanoids is not always predictable due to unique balances of context, products, and receptors.

Calpain 1 cleaves and inactivates prostacyclin synthase in mesenteric arteries from diabetic mice.

Diabetes is associated with a number of co-morbidities including an increased risk of developing cardiovascular diseases. The activation of Ca2+-activated proteases of the calpain family has been implicated in platelet activation associated with diabetes and this study aimed to determine the role of calpain activation in the development of endothelial dysfunction. Diabetes induction in miceattenuated acetylcholine-induced relaxation of mesenteric artery rings, an effect prevented in mice receiving a calpain inhibitor. A nitric oxide-independent but diclofenac-sensitive component of the relaxation-response was altered and correlated with a loss of prostacyclin (PGI2) generation and reduced vascular levels of PGI2 synthase. Calpain inhibition was also able to restore PGI2 synthase levels and PGI2 generation in arteries from diabetic animals. The effects of diabetes were reproduced in vitro by a combination of high glucose and palmitate, which elicited calpain activation, PGI2 synthase cleavage and inactivation as well as endothelial dysfunction in mesenteric arteries from wild-type mice. PGI2 cleavage was not observed in arteries from calpain 1-/- mice or mice overexpressing the endogenous calpain inhibitor calpastatin. Finally, proteomic analyses revealed that calpain 1 cleaved the C-terminal domain of PGI2 synthase close to the catalytic site of the enzyme. These data demonstrate that diabetes leads to the activation of calpain 1 in mesenteric arteries and can initiate endothelial dysfunction by cleaving and inactivating the PGI2 synthase. Given that calpain inhibition prevented diabetes-induced endothelial dysfunction in mesenteric arteries, calpains represent an interesting therapeutic target for the prevention of cardiovascular complication of diabetes.

Rosuvastatin inhibits pressure-induced fibrotic responses via the expression regulation of prostacyclin and prostaglandin E2 in rat renal tubular cells

Statins are reported to alleviate renal fibrosis in animal models with ureteral obstruction. However, the molecular mechanism of this antifibrotic effect is still unclear. Pressure force is an important mechanism contributing to induction and progression of tubulointerstitial fibrogenesis in ureteric obstruction. In this study, we investigated the influence of rosuvastatin on pressure-induced fibrotic responses in rat renal tubular cells (NRK-52E). We established an in vitro pressure culture system to study pressure-induced fibrotic responses in NRK-52E cells. When NRK-52E cells were cultured in the pressure culture system, 60mmHg of pressure induced the expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)-β, fibronectin, Smad3, and phospho-Smad3. Rosuvastatin significantly reduced these pressure-induced fibrotic responses at concentrations above 10μM. Rosuvastatin also reduced the TGF-β-induced expression of fibronectin and CTGF in NRK-52E cells. Pretreatment with rosuvastatin significantly induced prostacyclin (PGI2) generation, but reduced pressure-induced prostaglandin E2 (PGE2). PGI2 synthase small interfering RNA (siRNA) transfection significantly inhibited rosuvastatin-induced peroxisome proliferator-activated receptor α activation. The blockage of peroxisome proliferator-activated receptor α by siRNA transfection reduced the inhibitory effect of rosuvastatin on pressure-induced fibrotic responses. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398), a specific inhibitor of cyclooxygenase-2, diminished pressure-induced PGE2 generation, and also reduced pressure-induced fibrotic responses. Additionally, PGE2 decreased the antifibrotic effect of rosuvastatin. In conclusion, rosuvastatin reduces pressure-induced fibrotic responses in renal tubular cells by enhancing the PGI2-peroxisome proliferator-activated receptor α pathway and reducing PGE2 generation.

Intracellular free calcium content plays a role in low magnesium-induced increases in prostacyclin production

In a previous study, we found that magnesium deficiency stimulated prostacyclin production and suggested that this stimulation resulted from an enhanced Ca2+ influx induced by magnesium deficiency. In this study, we further examined prostacyclin generation in cultured human umbilical vein endothelial cells after intracellular free calcium content ([Ca2+]i) was altered by addition of diltiazem, nifedipine, verapamil, sodium cyanide (NaCN), ruthenium red or quinidine to a low magnesium medium. The results showed that diltiazem, nifedipine, verapamil and ruthenium red inhibited 45Ca2+ influx, and NaCN and quinidine had no effect on 45Ca2+ influx. However, all of these compounds decreased [Ca2+]i, [3H]arachidonic acid release and prostacyclin production. The reduced [3H]arachidonic acid content in cellular phospholipids caused by low magnesium treatment was not altered by the added compounds. We suggested that arachidonic acid release and prostacyclin production was calcium-dependent in cultured endothelial cells.

Abstract 8987: Prostacyclin Reduction Upregulates Tissue Factor and Predisposes COX-2 Knockout Mice to Thrombosis

Selective inhibitors of cyclooxygenase-2 (COX-2) increase the risk of myocardial infarction and athero-thrombotic events, but the mechanisms responsible for these effects are not fully understood. To assess the role of COX-2 in arterial thrombosis, a well-studied model of ferric chloride-induced carotid artery injury was applied to COX-2 knockout (COX-2KO) mice and data were compared with those obtained in wild-type (WT) mice. We found that thrombus formation was significantly greater in COX-2-null compared to WT mice. We then explored the role of platelets in this model. Platelets isolated from blood of COX-2KO mice showed enhanced aggregation responses to collagen and ADP together with greater thromboxane production than WT platelets. Interestingly, cyclooxygenase-1 and thromboxane synthase protein levels were similar in both animal groups. In cross-transfusion experiments, we found that platelets from COX-2KO mice behaved similarly to WT platelets in terms of in vivo thrombus formation. Importantly, we observed that the activity of tissue factor (TF), initiator of blood coagulation, was elevated both in concentrated microparticles derived from plasma and in leukocytes or in the carotid arteries of COX-2KO mice. Furthermore, the arterial wall of COX-2KO mice expressed higher levels of TF mRNA compared to WT animals, and these levels correlated with both reduced prostacyclin synthase mRNA levels and decreased prostacyclin production. In addition, treatment with CA10441, a prostacyclin receptor (IP) antagonist, increased TF activity in WT mice. Conversely, carbaprostacyclin, a stable IP agonist, reduced TF activity in COX-2KO mice. Overall, these findings reveal for the first time that the propensity to thrombosis observed in association with COX-2 inhibition is consequent to the impairment in the generation of prostacyclin by vascular endothelium, which in turn results in increased TF expression and activity.

Functions of Platelet Microparticles in Inflammatory Arthritis

Abstract SCI-35Besides their pivotal role in thrombosis and wound repair, platelets can participate in inflammatory responses via a broad arsenal that includes CD40L and CD40, cyclooxygenase-1 mediated prostaglandins, leukotrienes, IL-1, RANTES, serotonin, platelet factor 4, matrix metalloproteinases -2 and -9, P-selectin, adenosine diphosphate, and reactive oxygen species. Rheumatoid arthritis (RA) is amongst the most common autoimmune chronic inflammation that affects the joints. Interestingly, we found copious amounts of submicron particles that harbor platelet integrins in the synovial fluid of patients suffering from RA, pointing to evidence of platelet activation in this inflammatory autoimmune disease. Importantly, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. In addition to the transcellular collaboration between platelets and fibroblast-like synoviocytes in generation of pro-inflammatory prostacyclin (1), we found that platelets contribute to synovitis by production of IL-1-rich platelet microparticles (2). These recent advances in understanding of the platelet activities in inflammation notwithstanding, the mechanisms by which platelet microparticles invade the diseased joint in RA remain obscure. Using synovial biopsies from patients suffering from RA in addition to in vivo imaging strategies in a murine model of arthritis, our current work aspires to dissect the means of transportation of platelet microparticles during RA. Given their pro-inflammatory properties, to understand the process by which microparticles invade the synovial joint is of great clinical interest. Disclosures:No relevant conflicts of interest to declare.

Long-lasting inhibition of platelet prostaglandin but normal vascular prostacyclin generation following sulphinpyrazone administration to rats.

Long-lasting inhibition of platelet prostaglandin but normal vascular prostacyclin generation following sulphinpyrazone administration to rats.

Platelets Participate in Synovitis via Cox-1–Dependent Synthesis of Prostacyclin Independently of Microparticle Generation

In addition to the well-described role of platelets in thrombosis, a growing body of evidence implicates platelets in diverse inflammatory responses. We recently showed platelets can contribute to the pathophysiology of inflammatory arthritis via IL-1- containing microparticles. In this study, we demonstrate that platelets, and not platelet microparticles, actively contribute to synovitis via production of proinflammatory prostacyclin in an autoimmune arthritis model. Using both genetic and pharmacologic approaches, we establish that paracrine production of prostacyclin proceeds in the absence of cyclooxygenase-2. Furthermore, we also demonstrate that prostacyclin generation can arise via transcellular collaboration between platelets and fibroblast-like synoviocytes. In addition to shedding light on an unappreciated pathway of lipid synthesis in arthritis, we further delineate a novel effector activity by which platelets can contribute to inflammatory disease.

Lymphocyte responses exacerbate angiotensin II-dependent hypertension

Activation of the immune system by ANG II contributes to the pathogenesis of hypertension, and pharmacological suppression of lymphocyte responses can ameliorate hypertensive end-organ damage. Therefore, to examine the mechanisms through which lymphocytes mediate blood pressure elevation, we studied ANG II-dependent hypertension in scid mice lacking lymphocyte responses and wild-type controls. Scid mice had a blunted hypertensive response to chronic ANG II infusion and accordingly developed less cardiac hypertrophy. Moreover, lymphocyte deficiency led to significant reductions in heart and kidney injury following 4 wk of angiotensin. The muted hypertensive response in the scid mice was associated with increased sodium excretion, urine volumes, and weight loss beginning on day 5 of angiotensin infusion. To explore the mechanisms underlying alterations in blood pressure and renal sodium handling, we measured gene expression for vasoactive mediators in the kidney after 4 wk of ANG II administration. Scid mice and controls had similar renal expression for interferon-gamma, interleukin-1beta, and interleukin-6. By contrast, lymphocyte deficiency (i.e., scid mice) during ANG II infusion led to upregulation of tumor necrosis factor-alpha, endothelial nitric oxide synthase (eNOS), and cyclooxygenase-2 (COX-2) in the kidney. In turn, this enhanced eNOS and COX-2 expression in the scid kidneys was associated with exaggerated renal generation of nitric oxide, prostaglandin E(2), and prostacyclin, all of which promote natriuresis. Thus, the absence of lymphocyte activity protects from hypertension by allowing blood pressure-induced sodium excretion, possibly via stimulation of eNOS- and COX-2-dependent pathways.

Kallikrein Inhibitors as Antiinflammatory Therapeutics.

Abstract 3179Poster Board III-118Plasma kallikrein is a novel target for antiinflammatory inhibitor design since it plays a role in the initiation of the intrinsic clotting, fibrinolysis, bradykinin-forming and neutrophil activation. The goal of this study was to synthesize a potent and selective inhibitor of kallikrein for the prevention and/or treatment of hyperfibrinolytic states, systemic inflammation, sepsis as well as cardiopulmonary bypass. Using a focus library of compounds, UK-097820, CP-013118 and PF-04886847 were found not to be cytotoxic and had the ability to abolish the hydrolysis of S2302 by kallikrein both in fluid phase and on the surface-mediated activation. In a competition study, PF-04886847 (3 mM) reduced the formation of a complex between C1-inhibitor and kallikrein by 40% following the activation of prekallikrein (kallikrein zymogen) on bound high molecular weight kininogen (HK) to human pulmonary artery endothelial cell (HPAEC). It was also mechanistically important to distinguish between direct kallikrein inhibition caused by the binding of kallikrein inhibitor to kallikrein versus the inhibitor's indirect effect on HK, prekallikrein or perturbation of the membrane function. The binding of PK to HK or HK to HPAEC surface was unaffected by the kallikrein inhibitors. To determine the selectivities of the kallikrein inhibitors, the effects of UK-097820, CP-013118 and PF-04886847-00 on factor XI and factor XII were examined. PF-04886847 was found to be a potent and selective inhibitor of kallikrein at a concentration ranging from 1 to 3 mM. BK stimulates cultured endothelial cells to release various mediators. The effects of the kallikrein inhibitors on the generation of bradykinin (BK), nitric oxide (NO) and prostacyclin and intracellular calcium signaling were determined. The generation of BK, NO formation and 6-keto-PGF1a release were blocked by PF-04886847, indicating that PF-04886847 might have potential applications for controlling unwanted inflammation provoked by BK-induced endothelium activation. In situ study, PF-04886847 blocked PK activation on intact aorta precontracted by phenylephrine. Taken together, we propose that PF-04886847 treatment might be beneficial in tissue injury and have potential clinical application. DisclosuresNo relevant conflicts of interest to declare.

Coupling factor 6-induced prostacyclin inhibition is enhanced in vascular smooth muscle cells from spontaneously hypertensive rats

Coupling factor 6 (CF6) attenuates the endothelial generation of prostacyclin. However, the role of CF6 in the resistance arteriole that is directly related to vascular tone is not determined yet. We investigated the effect of endogenous and exogenous CF6 on prostacyclin generation in cultured vascular smooth muscle cells (VSMCs). We cultured resistance arteriole VSMCs from the mesenteric artery network of spontaneously hypertensive rats (SHRs, n = 8) and Wistar-Kyoto rats (WKY, n = 8) by enzymatic method. The gene expression of CF6 was higher by 76 +/- 24% in SHR-derived VSMCs compared with WKY rat-derived VSMCs (P < 0.05) concomitant with the reduced degradation rate of CF6 mRNA. The release of CF6 in SHRs was higher than that in WKY rats (11.0 +/- 0.8 vs. 3.8 +/- 0.4 pg/microg protein, P < 0.05). Prostacyclin generation was attenuated in mesenteric arteriolar VSMCs from SHRs compared with those from WKY rats, but it was restored by neutralization of CF6 with its antibody. Exogenous administration of CF6 suppressed arachidonic acid release in a dose-dependent manner, and it was greater in SHRs than in WKY rats. Pretreatment with PP1, an inhibitor of tyrosine kinase c-Src, or receptor blockers such as ADP, efrapeptin, and an antibody to beta-subunit of ATP synthase blocked CF6-induced decrease in prostacyclin generation. These data suggest that CF6 suppresses prostacyclin generation in resistance arteriole VSMCs in an autocrine or paracrine fashion, and it is enhanced in SHRs by the overproduction of CF6 and the hyperresponsiveness to CF6.

High-density Lipoprotein Cholesterol Levels and Prognosis in Advanced Heart Failure

High-density lipoproteins (HDLs) influence the generation of prostacyclin via cyclooxygenase stimulation. Prostaglandins represent an important compensatory pathway in advanced heart failure (HF). Whether HDL levels discriminate prognosis in HF remains unknown. We prospectively evaluated the prognostic relationship of HDL levels in severe HF by examining 132 consecutive patients listed for heart transplantation (52 +/- 11 years of age, 80% men, 79% white, mean follow-up 18 months). Using population mean HDL levels (HDL <33 mg/dl [n = 47] vs > or =33 mg/dl [n = 85]), patients were grouped and followed for the primary composite end-points of HF hospitalizations or death, stratified by underlying etiology (non-ischemic, n = 52; ischemic, n = 80). Patients with HDL <33 mg/dl had lower serum sodium (135 vs 137 mEq/liter, p = 0.008), higher total bilirubin (1.3 vs 0.7 mg/dl, p < 0.001) and higher uric acid (7.6 vs 6.7 mg/dl, p = 0.048) levels, but similar serum creatinine compared with the > or =33 mg/dl HDL group. Survival analysis, using a Cox proportional hazards model, revealed reduced HDL (<33 mg/dl) as the most significant independent predictor of HF hospitalizations or death, independent of underlying etiology. Low-cholesterol and low-density lipoprotein (LDL)-cholesterol alone were not found to be independently predictive of outcome. Lower HDL levels correlate with adverse prognosis independent of etiology and predict clinical worsening or death in advanced HF. Further study is warranted as to whether these findings represent a clinical marker or suggest a potential therapeutic target.

Curcumin prevents the oxidation and lipid modification of LDL and its inhibition of prostacyclin generation by endothelial cells in culture

Low-density lipoprotein (LDL) was isolated from human plasma and oxidized by 5 μM copper sulfate for 4 h at 37 °C in the absence and presence of 1, 3, 5, 10, or 20 μM of curcumin. LDL oxidized in the absence of curcumin (oxLDL) showed an increased levels of conjugated dienes, lipid peroxides (TBARS) and lysolecithin (lysoPC) and a significant loss of polyunsaturated fatty acids (PUFA). LDL oxidized with 5 μM copper sulfate in the presence of curcumin caused a significant decrease of conjugated diene, lipid peroxides, lysoPC and significant increase of PUFA compared to oxLDL. These changes were dose dependent and reached a maximum at 5 μM curcumin. Incubation of human endothelial cells (EC) with 200 μg protein/ml of oxLDL caused a significant decrease of prostacyclin (PGI 2) generation. LDL oxidized in presence of 5 μM curcumin did not show any inhibition of PGI 2 generation compared to the control cells. These results indicate that curcumin is an effective chain-breaking antioxidant which prevents oxidation and lipid modification of LDL. The inhibition of oxLDL on PGI 2 is considered a contributing factor in the pathogenesis of thrombosis and atherosclerosis. Curcumin supplementation could be an effective strategy in preventing LDL oxidation and its impact on atherosclerosis and lesion formation.

Peroxisome proliferator-activated receptor alpha plays a crucial role in L-carnitine anti-apoptosis effect in renal tubular cells

L-carnitine is synthesized mainly in the liver and kidneys from lysine and methionine from dietary sources. Many reports have shown that L-carnitine can protect certain cells against the toxicity of several anticancer and toxic agents, although the detailed mechanism is poorly understood. In this study, we investigated the protective effect of L-carnitine and its molecular mechanism in renal tubular cells undergoing gentamicin-induced apoptosis. Rat tubular cell line (NRK-52E) and mice were used as the model system. Gentamicin-induced apoptosis in renal tubular cells was examined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling. We introduced short interfering RNA transfection and gene-deficient mice to investigate the protective mechanism of L-carnitine. We found that L-carnitine inhibited gentamicin-induced reactive oxygen species generation and correlative apoptotic pathways, resulting in the protection of NRK-52E cells from gentamicin-induced apoptosis. The treatment of L-carnitine also lessened gentamicin-induced renal tubular cell apoptosis in mice. L-carnitine was found to increase the prostacyclin (PGI(2)) generation in NRK-52E cells. The siRNA transfection for PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and L-carnitine's protective effect. We found that the activity of the potential PGI(2) nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), was elevated by L-carnitine treatment. The siRNA-mediated blockage of PPARalpha considerably reduced the anti-apoptotic effect of L-carnitine. In PPARalpha-deficient mice, L-carnitine treatment also lost the inhibitory effect on gentamicin-induced apoptosis in kidneys. Based on these findings, we suggest that L-carnitine protects renal tubular cells from gentamicin-induced apoptosis through PGI(2)-mediated PPARalpha activation.

Coupling factor 6 as a novel vasoactive and proatherogenic peptide in vascular endothelial cells

Coupling factor 6 (CF6) is composed of 76 amino acids and is present in the peripheral stalk of mitochondrial ATP synthase. The generation of CF6 is positively regulated by tumor necrosis factor alpha and shear stress via nuclear factor kappaB, and by high glucose via protein kinase C and p38 mitogen-activated protein kinase. CF6 is released outside of the cells from vascular endothelial cells, and binds to the beta-subunit of the plasma membrane-bound ATP synthase in vascular endothelial cells and leads to intracellular acidosis. CF6 produces vasoconstriction, and the biological active site resides at the C-terminal portion. CF6 suppresses prostacyclin generation via inhibition of cytosolic phospholipase A(2). CF6 also suppresses nitric oxide synthase activity via an increase in asymmetric dimethylarginine and a decrease in platelet/endothelial cell adhesion molecule-1. CF6 induces the gene and protein expression of proatherogenic molecules such as endothelin 2, urokinase type plasminogen activator receptor, estrogen receptor beta, a soluble short form of vascular endothelial growth factor receptor-1, and lectin-like oxidized low-density lipoprotein receptor-1. The plasma level of CF6 is elevated in patients with essential hypertension, diabetes mellitus, end-stage renal disease, acute myocardial infarction, and coronary heart disease. It is likely that CF6 contributes to the pathogenesis of cardiovascular diseases, but further intensive investigation is needed.

L-Carnitine attenuates angiotensin II-induced proliferation of cardiac fibroblasts: role of NADPH oxidase inhibition and decreased sphingosine-1-phosphate generation

The heart is unable to synthesize l-carnitine and is strictly dependent on the l-carnitine provided by the blood stream; however, additional studies are needed to better understand the mechanism of l-carnitine supplementation to the heart. The aim of this study was to evaluate the effects of l-carnitine on angiotensin II (Ang II)-induced cardiac fibroblast proliferation and to explore its intracellular mechanism(s). Cultured rat cardiac fibroblasts were pretreated with l-carnitine (1-30 mM) then stimulated with Ang II (100 nM). Ang II increased fibroblast proliferation and endothelin-1 expression, which were partially inhibited by l-carnitine. l-Carnitine also attenuated Ang II-induced NADPH oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, activator protein-1-mediated reporter activity and sphingosine-1-phosphate generation. In addition, l-carnitine increased prostacyclin (PGI 2) generation in cardiac fibroblasts. siRNA transfection of PGI 2 synthase significantly reduced l-carnitine-induced PGI 2 and its anti-proliferation effects on cardiac fibroblasts. Furthermore, blockading potential PGI 2 receptors, including immunoprecipitation (IP) receptors and peroxisome proliferator-activated receptors alpha (PPARα) and delta , revealed that siRNA-mediated blockage of PPARα considerably reduced the anti-proliferation effect of l-carnitine. In summary, these results suggest that l-carnitine attenuates Ang II-induced effects (including NADPH oxidase activation, sphingosine-1-phosphate generation and cell proliferation) in part through PGI 2 and PPARα-signaling pathways.