Sven Horke

Vascular oxidative stress, nitric oxide and atherosclerosis

In the vascular wall, reactive oxygen species (ROS) are produced by several enzyme systems including NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase (eNOS) and the mitochondrial electron transport chain. On the other hand, the vasculature is protected by antioxidant enzyme systems, including superoxide dismutases, catalase, glutathione peroxidases and paraoxonases, which detoxify ROS. Cardiovascular risk factors such as hypercholesterolemia, hypertension, and diabetes mellitus enhance ROS generation, resulting in oxidative stress. This leads to oxidative modification of lipoproteins and phospholipids, mechanisms that contribute to atherogenesis. In addition, oxidation of tetrahydrobiopterin may cause eNOS uncoupling and thus potentiation of oxidative stress and reduction of eNOS-derived NO, which is a protective principle in the vasculature. This review summarizes the latest advances in the role of ROS-producing enzymes, antioxidative enzymes as well as NO synthases in the initiation and development of atherosclerosis.

Paraoxonase-2 Reduces Oxidative Stress in Vascular Cells and Decreases Endoplasmic Reticulum Stress–Induced Caspase Activation

Background— In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response. Methods and Results— Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we show that overexpression of PON2 markedly reduced ROS, whereas its knockdown increased ROS levels significantly. Using microscopic and biochemical methods, we found PON2 mainly in the nuclear membrane and endoplasmic reticulum. Furthermore, PON2 expression was induced at both the promoter and protein levels by endoplasmic reticulum stress pathway unfolded protein response. This pathway may promote both apoptotic and survival mechanisms. Functionally, PON2 reduced unfolded protein response–accompanying oxidative stress and unfolded protein response–derived caspase activation. Conclusion— We suggest that PON2 represents an endogenous defense mechanism against vascular oxidative stress and unfolded protein response–induced cell death, thereby contributing to the prevention of atherosclerosis.

Oxidative stress in vascular disease and its pharmacological prevention.

Cardiovascular risk factors lead to enhanced production of reactive oxygen species (ROS) generated by NADPH oxidase, xanthine oxidase (XO), the mitochondrial electron-transport chain (ETC), and dysfunctional endothelial nitric oxide synthase (eNOS). When the capacity of antioxidant defense systems [e.g., superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), heme oxygenase (HO), paraoxonase (PON)] is exceeded, this results in oxidative stress, which can promote atherogenesis. Therefore, pharmacological means to prevent oxidative stress are of major therapeutic interest. Some established drugs and novel therapeutic approaches can prevent oxidative stress and, presumably, vascular disease. These include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor type 1 (AT1 receptor) blockers (ARBs), statins, nebivolol, pentaerithrityl tetranitrate (PETN), resveratrol, and mitochondria-targeted antioxidants. Molecular mechanisms involved in the induction of oxidative stress under pathological conditions as well as pharmacological approaches (and their molecular mechanisms) are summarized in this review.

Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N-(3-Oxododecanoyl)-L-Homoserine Lactone

ABSTRACT The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-Oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosas quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 ≫ PON1192R > PON1192Q > PON3. PON2 exhibited a high specific activity of 7.6 ± 0.4 μmols/min/mg at 10 μM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

One Enzyme, Two Functions PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O2̇̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O2̇̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His114 and His133 are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys311, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.

Differential roles of PKCα and PKCɛ in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxide. PMA activated the PKC isoforms alpha and epsilon in HUVEC and EA.hy 926 cells. Knockdown of PKCepsilon by siRNA prevented the early downregulation of Nox4, whereas knockdown of PKCalpha selectively abolished the late Nox4 upregulation. Vascular endothelial growth factor (VEGF), which activates PKCalpha but not PKCepsilon in HUVEC, increased Nox4 expression without the initial downregulation. VEGF-induced Nox4 upregulation was associated with an enhanced proliferation and angiogenesis of HUVEC. Both effects could be reduced by inhibition of NADPH oxidase. Thus, a selective inhibition/knockdown of PKCalpha may represent a novel therapeutic strategy for vascular disease.

Nuclear Trafficking of La Protein Depends on a Newly Identified Nucleolar Localization Signal and the Ability to Bind RNA

Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments.

Reciprocal Regulation of Endothelial Nitric-Oxide Synthase and NADPH Oxidase by Betulinic Acid in Human Endothelial Cells

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective principle in the vasculature. Many cardiovascular diseases are associated with reduced NO bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA and protein expression, and NO production in a concentration- and time-dependent manner. Major ZSS constituents include saponins, such as jujuboside A and B, and pentacyclic triterpenes, such as betulin and betulinic acid. Jujuboside A, jujuboside B, or betulin had no significant effect on eNOS expression, whereas betulinic acid increased eNOS mRNA and protein expression in HUVEC and EA.hy 926 cells. Interestingly, betulinic acid also attenuated the expression of NADPH oxidase subunits Nox4 and p22phox, thereby reducing oxidative stress and improving eNOS function. Consequently, betulinic acid-treated endothelial cells showed an increased production of bioactive NO (as indicated by a higher efficacy in stimulating cGMP generation in RFL-6 reporter cells). Thus, betulinic acid possesses combined properties of eNOS up-regulation and NADPH oxidase down-regulation. Compounds such as betulinic acid may have a therapeutic potential in cardiovascular disease.

Uncoupling of Endothelial Nitric Oxide Synthase in Perivascular Adipose Tissue of Diet-Induced Obese Mice

Objective— The present study was conducted to investigate the contribution of perivascular adipose tissue (PVAT) to vascular dysfunction in a mouse model of diet-induced obesity. Approach and Results— Obesity was induced in male C57BL/6J mice with a high-fat diet for 20 weeks, and vascular function was studied with myograph. In PVAT-free aortas isolated from obese mice, the endothelium-dependent, nitric oxide–mediated vasodilator response to acetylcholine remained normal. In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when PVAT was left in place. Adipocytes in PVAT were clearly positive in endothelial nitric oxide synthase (eNOS) staining, and PVAT nitric oxide production was significantly reduced in obese mice. High-fat diet had no effect on eNOS expression but led to eNOS uncoupling, evidenced by diminished superoxide production in PVAT after eNOS inhibition. As mechanisms for eNOS uncoupling, arginase induction and L-arginine deficiency were observed in PVAT. Obesity-induced vascular dysfunction could be reversed by ex vivo L-arginine treatment and arginase inhibition. Conclusions— Diet-induced obesity leads to L-arginine deficiency and eNOS uncoupling in PVAT. The combination therapy with L-arginine and arginase inhibitors may represent a novel therapeutic strategy for obesity-induced vascular disease.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3s anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.