Biomarkers in ROS and Role of Isoprostanes in Oxidative Stress

Biomarkers of reactive oxygen species serve as indicators of oxidative stress in the pathology of cardiovascular diseases. This chapter presents an overview of the various biomarkers available to quantify oxidative stress to advance the understanding of the pathophysiology of cardiovascular diseases as well as to serve as an adjunct in their diagnosis and prognosis. The plasma levels of reactive oxygen species themselves are unstable and unreliable markers of oxidative stress. The commonly used stable biomarkers are derivatives of oxygen radicals such as products of lipid peroxidation and protein oxidation, with isoprostanes and malondialdehyde (MDA) being the most widely used biomarkers due to higher specificity and ease of measurement. Recently, micro‐RNA is emerging as stable and specific biomarkers for detection of heart failure. Other biomarkers have a role in certain conditions; for example, advanced oxidation protein products indicate acute inflammation, whereas advanced glycation end products serve as indicators of chronic disease

PPAR γ

Peroxisome proliferator-activated receptor Gamma (PPARγ), a ligand-activated transcription factor, has a role in various cellular functions as well as glucose homeostasis, lipid metabolism, and prevention of oxidative stress. The activators of PPARγ are already widely used in the treatment of diabetes mellitus. The cardioprotective effect of PPARγ activation has been studied extensively over the years making them potential therapeutic targets in diseases associated with cardiovascular disorders. However, they are also associated with adverse cardiovascular events such as congestive heart failure and myocardial infarction. This review aims to discuss the role of PPARγ in the various cardiovascular diseases and summarize the current knowledge on PPARγ agonists from multiple clinical trials. Finally, we also review the new PPARγ agonists under development as potential therapeutics with reduced or no adverse effects

Synergistic Effect of Anemia and Red Blood Cells Transfusion on Inflammation and Lung Injury

Anemia and resultant red blood cell transfusion may be associated with adverse long-term clinical outcomes. To investigate the mechanism(s) responsible, we profiled inflammatory biomarkers and circulating levels of the bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) in control and anemic mice with or without LPS-induced systemic inflammation. Acute anemia or lipopolysaccharide (LPS) challenge alone triggered an increase of circulating levels of the inflammatory markers IL-6 and keratinocyte-derived chemokine (CXCL1/KC). Moreover, administration of LPS to anemic mice reduced circulating S1P levels and augmented lung injury and pulmonary vascular permeability. Transfusion of aged, but not fresh, red blood cells (RBCs) worsened pulmonary vascular leak. S1P levels decline markedly during storage of mouse RBCs. Loading stored murine RBCs with S1P prior to transfusion partially attenuated anemia-associated acute pulmonary vascular leak. Taken together, our results indicate that anemia and systemic inflammation can alter the S1P buffering capacity of RBCs, suggesting possible strategies for alleviating transfusion-related lung injury in clinical practice

Electrophilic Aldehyde 4-Hydroxy-2-Nonenal Mediated Signaling and Mitochondrial Dysfunction

Reactive oxygen species (ROS), a by-product of aerobic life, are highly reactive molecules with unpaired electrons. The excess of ROS leads to oxidative stress, instigating the peroxidation of polyunsaturated fatty acids (PUFA) in the lipid membrane through a free radical chain reaction and the formation of the most bioactive aldehyde, known as 4-hydroxynonenal (4-HNE). 4-HNE functions as a signaling molecule and toxic product and acts mainly by forming covalent adducts with nucleophilic functional groups in proteins, nucleic acids, and lipids. The mitochondria have been implicated as a site for 4-HNE generation and adduction. Several studies clarified how 4-HNE affects the mitochondria’s functions, including bioenergetics, calcium homeostasis, and mitochondrial dynamics. Our research group has shown that 4-HNE activates mitochondria apoptosis-inducing factor (AIFM2) translocation and facilitates apoptosis in mice and human heart tissue during anti-cancer treatment. Recently, we demonstrated that a deficiency of SOD2 in the conditional-specific cardiac knockout mouse increases ROS, and subsequent production of 4-HNE inside mitochondria leads to the adduction of several mitochondrial respiratory chain complex proteins. Moreover, we highlighted the physiological functions of HNE and discussed their relevance in human pathophysiology and current discoveries concerning 4-HNE effects on mitochondria

Cardiac-Specific Inactivation of LPP3 in Mice Leads to Myocardial Dysfunction and Heart Failure

Lipid Phosphate phosphatase 3 (LPP3), encoded by the Plpp3 gene, is an enzyme that dephosphorylates the bioactive lipid mediator lysophosphatidic acid (LPA). To study the role of LPP3 in the myocardium, we generated a cardiac specific Plpp3 deficient mouse strain. Although these mice were viable at birth in contrast to global Plpp3 knockout mice, they showed increased mortality ~ 8 months. LPP3 deficient mice had enlarged hearts with reduced left ventricular performance as seen by echocardiography. Cardiac specific Plpp3 deficient mice had longer ventricular effective refractory periods compared to their Plpp3 littermates. We observed that lack of Lpp3 enhanced cardiomyocyte hypertrophy based on analysis of cell surface area. We found that lack of Lpp3 signaling was mediated through the activation of Rho and phospho-ERK pathways. There are increased levels of fetal genes Natriuretic Peptide A and B (Nppa and Nppb) expression indicating myocardial dysfunction. These mice also demonstrate mitochondrial dysfunction as evidenced by a significant decrease (P \u3c 0.001) in the basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity as measured through mitochondrial bioenergetics. Histology and transmission electron microscopy of these hearts showed disrupted sarcomere organization and intercalated disc, with a prominent disruption of the cristae and vacuole formation in the mitochondria. Our findings suggest that LPA/LPP3-signaling nexus plays an important role in normal function of cardiomyocytes

SOD2 Deficiency in Cardiomyocytes Defines Defective Mitochondrial Bioenergetics as a Cause of Lethal Dilated Cardiomyopathy

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy

Lysophosphatidic Acid Receptors 1 and 2 Play Roles in Regulation of Vascular Injury Responses but Not Blood Pressure

Phenotypic modulation of vascular smooth muscle cells (SMC) is essential for the development of intimal hyperplasia. Lysophosphatidic acid (LPA) is a serum component that can promote phenotypic modulation of cultured SMC, but an endogenous role for this bioactive lipid as a regulator of SMC function in vivo has not been established. Ligation injury of the carotid artery in mice increased levels in the vessel of both autotaxin, the lysophospholipaseD enzyme responsible for generation of extracellular LPA, and two LPA responsive G-protein coupled receptors 1 (LPA1) and 2 (LPA2). LPA1-/-2-/- mice were partially protected from the development of injury-induced neointimal hyperplasia, whereas LPA1-/- mice developed larger neointimal lesions after injury. Growth in serum, LPA-induced ERK activation, and migration to LPA and serum were all attenuated in SMC isolated from LPA1-/-2-/- mice. In contrast, LPA1-/- SMCs exhibited enhanced migration resulting from an upregulation of LPA3. However, despite their involvement in intimal hyperplasia, neither LPA1 nor LPA2 were required for dedifferentiation of SMC following vascular injury or dedifferentiation of isolated SMC in response to LPA or serum in vitro. Similarly, neither LPA1 nor LPA2 were required for LPA to elicit a transient increase in blood pressure following intravenous administration of LPA to mice. These results identify a role for LPA and two defined LPA receptors in regulating SMC migratory responses in the context of vascular injury, but suggest that additional LPA receptor subtypes are required for other LPA-mediated effects in the vasculature

A Novel Tetrapeptide Derivative Exhibits In Vitro Inhibition of Neutrophil-Derived Reactive Oxygen Species and Lysosomal Enzymes Release

Neutrophil infiltration plays a major role in the pathogenesis of myocardial injury. Oxidative injury is suggested to be a central mechanism of the cellular damage after acute myocardial infarction. This study is pertained to the prognostic role of a tetrapeptide derivative PEP1261 (BOC-Lys(BOC)-Arg-Asp-Ser(tBu)-OtBU), a peptide sequence (39–42) of lactoferrin, studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation, lysosomal enzymes release, and enhanced expression of C proteins. The groundwork experimentation was concerned with the isolation of neutrophils from the normal and acute myocardial infarct rats to find out the efficacy of PEP1261 in the presence of a powerful neutrophil stimulant, phorbol 12-myristate 13 acetate (PMA). Stimulation of neutrophils with PMA resulted in an oxidative burst of superoxide anion and enhanced release of lysosomal enzymes and expression of complement proteins. The present study further demonstrated that the free radicals increase the complement factors in the neutrophils confirming the role of ROS. PEP1261 treatment significantly reduced the levels of superoxide anion and inhibited the release of lysosomal enzymes in the stimulated control and infarct rat neutrophils. This study demonstrated that PEP1261 significantly inhibited the effect on the ROS generation as well as the mRNA synthesis and expression of the complement factors in neutrophils isolated from infarct heart

Protective Effect of Low-Dose Alcohol Consumption against Post-Ischemic Neuronal Apoptosis: Role of L-PGDS

Ischemic stroke is one of the leading causes of permanent disability and death in adults worldwide. Apoptosis is a major element contributing to post-ischemic neuronal death. We previously found that low-dose alcohol consumption (LAC) protects against neuronal apoptosis in the peri-infarct cortex following transient focal cerebral ischemia. Lipocalin-type prostaglandin D2 synthase (L-PGDS), which is mainly localized in the central nervous system (CNS), was previously shown to inhibit neuronal apoptosis. Therefore, we determined whether L-PGDS is involved in the protective effect of LAC against post-ischemic neuronal apoptosis. Wild-type (WT), CaMKIIαCreERT2/+/L-PGDS+/+, and CaMKIIαCreERT2/+/L-PGDSflox/flox mice on a C57BL/6J background were gavage fed with ethanol or volume-matched water once a day for 8 weeks. Tamoxifen (2 mg/day) was given intraperitoneally to CaMKIIαCreERT2/+/L-PGDS+/+ and CaMKIIαCreERT2/+/L-PGDSflox/flox mice for 5 days during the fourth week. AT-56 (30 mg/kg/day), a selective inhibitor of L-PGDS, was given orally to AT-56-treated WT mice from the fifth week for four weeks. Cerebral ischemia/reperfusion (I/R) injury, TUNEL-positive neurons, and cleaved caspase-3-positive neurons were measured at 24 h of reperfusion after a 90 min unilateral middle cerebral artery occlusion (MCAO). We found that 0.7 g/kg/day but not 2.8 g/kg/day ethanol significantly upregulated L-PGDS in the cerebral cortex. In addition, 0.7 g/kg/day ethanol diminished cerebral ischemia/reperfusion (I/R) injury and TUNEL-positive and cleaved caspase-3-positive neurons in the peri-infarct cortex in WT and CaMKIIαCreERT2/+/L-PGDS+/+ mice. Furthermore, the neuroprotective effect of 0.7 g/kg/day ethanol was alleviated in AT-56-treated WT and CaMKIIαCreERT2/+/L-PGDSflox/flox mice. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing post-ischemic neuronal apoptosis via an upregulated L-PGDS