Role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in diabetic complications.

Cardiovascular disease (CVD) complications have remained a major cause of death among patients with diabetes. Hence, there is a need for effective therapeutics against diabetes-induced CVD complications. Since its discovery, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to be involved in the pathology of various CVDs, with studies showing a positive association between plasma levels of PCSK9, hyperglycemia, and dyslipidemia. PCSK9 regulates lipid homeostasis by interacting with low-density lipoprotein receptors (LDLRs) present in hepatocytes and subsequently induces LDLR degradation via receptor-mediated endocytosis, thereby reducing LDL uptake from circulation. In addition, PCSK9 also induces pro-inflammatory cytokine expression and apoptotic cell death in diabetic-CVD. Furthermore, therapies designed to inhibit PCSK9 effectively reduces diabetic dyslipidemia with clinical studies reporting reduced cardiovascular events in patients with diabetes and no significant adverse effect on glycemic controls. In this review, we discuss the role of PCSK9 in the pathogenesis of diabetes-induced CVD and the potential mechanisms by which PCSK9 inhibition reduces cardiovascular events in diabetic patients.

Mechanisms of COVID-19 pathogenesis in diabetes.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a global pandemic impacting 254 million people in 190 countries. Comorbidities, particularly cardiovascular disease, diabetes, and hypertension, increase the risk of infection and poor outcomes. SARS-CoV-2 enters host cells through the angiotensin-converting enzyme-2 receptor, generating inflammation and cytokine storm, often resulting in multiorgan failure. The mechanisms and effects of COVID-19 on patients with high-risk diabetes are not yet completely understood. In this review, we discuss the variety of coronaviruses, structure of SARS-CoV-2, mutations in SARS-CoV-2 spike proteins, receptors associated with viral host entry, and disease progression. Furthermore, we focus on possible mechanisms of SARS-CoV-2 in diabetes, leading to inflammation and heart failure. Finally, we discuss existing therapeutic approaches, unanswered questions, and future directions.

Preparation of LDL , Oxidation , Methods of Detection, and Applications in Atherosclerosis Research.

The concept of lipid peroxidation has been known for a long time. It is now well established that LDL plays a major role in atherosclerosis. Oxidized low-density lipoprotein (Ox-LDL) has been studied for over 35 years. Numerous pro- and anti-atherogenic properties have been attributed to Ox-LDL. Component composition of Ox-LDL is complex due to the influence of various factors, including the source, method of preparation, storage and use. Hence, it is very difficult to clearly define and characterize Ox-LDL. It contains unoxidized and oxidized fatty acid derivatives both in the ester and free forms, their decomposition products, cholesterol and its oxidized products, proteins with oxidized amino acids and cross-links, polypeptides with varying extents of covalent modification with lipid oxidation products and many others. The measurement of lipid oxidation has been a great boon, not only to the understanding of the process but also in providing numerous serendipitous discoveries and methodologies. In this chapter, we outline the methodologies for the preparation and testing of various lipoproteins for oxidation studies.

Doxorubicin-induced apoptosis enhances monocyte infiltration and adverse cardiac remodeling in diabetic animals.

Diabetic cancer patients were treated with doxorubicin (DOX), a potent chemotherapeutic drug that induces cardiac toxicity; however, molecular mechanisms of cardiac toxicity in this specific disease progression in patients and animal models are completely unknown. Therefore, we designed a study to understand the effects of DOX-induced cardiac toxicity in diabetic animals and the involved pathophysiological mechanisms. C57BL/6 J mice were divided into four DOX- and diabetic (streptozotocin; STZ) - treated groups; control, STZ, DOX, and DOX+STZ. At day 14, animals were sacrificed, echocardiography was used to examine heart function, and heart and blood samples were collected to investigate apoptotic mechanisms (caspase 3, BAX, B-Cell leukemia/lymphoma 2 (Bcl2)), inflammation, and cardiac remodeling. Our data shows a significant (p < 0.05) increase in glucose levels, apoptotic markers, and monocyte infiltration at the site of apoptosis and triggered inflammatory immune response (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)), in DOX+STZ animals compared with control and experimental groups. We also observed significant (p < 0.05) increase in myofibrillar area, fibrosis, and significantly decreased (p < 0.05) cardiac function in DOX-treated diabetic animals compared with controls. In conclusion, our data suggest that DOX induces significantly increased apoptosis, fibrosis, and structural alterations in diabetic hearts compared with non-diabetic animals.

Amelioration of diabetes-induced inflammation mediated pyroptosis, sarcopenia, and adverse muscle remodelling by bone morphogenetic protein-7.

BACKGROUND: Diabetic myopathy involves hyperglycaemia and inflammation that causes skeletal muscle dysfunction; however, the potential cellular mechanisms that occur between hyperglycaemia and inflammation, which induces sarcopenia, and muscle dysfunction remain unknown. In this study, we investigated hyperglycaemia-induced inflammation mediating high-mobility group box 1 activation, which is involved in a novel form of cell death, pyroptosis, diabetic sarcopenia, atrophy, and adverse muscle remodelling. Furthermore, we investigated the therapeutic potential of bone morphogenetic protein-7 (BMP-7), an osteoporosis drug, to treat pyroptosis, and diabetic muscle myopathy. METHODS: C57BL6 mice were treated with saline (control), streptozotocin (STZ), or STZ + BMP-7 to generate diabetic muscle myopathy. Diabetes was established by determining the increased levels of glucose. Then, muscle function was examined, and animals were sacrificed. Gastrocnemius muscle or blood samples were analysed for inflammation, pyroptosis, weight loss, muscle atrophy, and adverse structural remodelling of gastrocnemius muscle using histology, enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and reverse transcription polymerase chain reaction. RESULTS: A significant (P < 0.05) increase in hyperglycaemia leads to an increase in inflammasome (high-mobility group box 1, toll-like receptor-4, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing protein 3) formation in diabetic muscle cells. Further analysis showed an up-regulation of the downstream pyroptotic pathway with significant (P < 0.05) number of positive muscle cells expressing pyroptosis-specific markers [caspase-1, interleukin (IL)-1ß, IL-18, and gasdermin-D]. Pyroptotic cell death is involved in further increasing inflammation by releasing pro-inflammatory cytokine IL-6. Structural analysis showed the loss of muscle weight, decreased myofibrillar area, and increased fibrosis leading to muscle dysfunction. Consistent with this finding, BMP-7 attenuated hyperglycaemia (~50%), pyroptosis, inflammation, and diabetic adverse structural modifications as well as improved muscle function. CONCLUSIONS: In conclusion, we report for the first time that increased hyperglycaemia and inflammation involve cellular pyroptosis that induces significant muscle cell loss and adverse remodelling in diabetic myopathy. We also report that targeting pyroptosis with BMP-7 improves diabetic muscle pathophysiology and muscle function. These findings suggest that BMP-7 could be a potential therapeutic option to treat diabetic myopathy.

The Role of Bone Morphogenetic Protein 7 (BMP-7) in Inflammation in Heart Diseases.

Bone morphogenetic protein-7 is (BMP-7) is a potent anti-inflammatory growth factor belonging to the Transforming Growth Factor Beta (TGF-ß) superfamily. It plays an important role in various biological processes, including embryogenesis, hematopoiesis, neurogenesis and skeletal morphogenesis. BMP-7 stimulates the target cells by binding to specific membrane-bound receptor BMPR 2 and transduces signals through mothers against decapentaplegic (Smads) and mitogen activated protein kinase (MAPK) pathways. To date, rhBMP-7 has been used clinically to induce the differentiation of mesenchymal stem cells bordering the bone fracture site into chondrocytes, osteoclasts, the formation of new bone via calcium deposition and to stimulate the repair of bone fracture. However, its use in cardiovascular diseases, such as atherosclerosis, myocardial infarction, and diabetic cardiomyopathy is currently being explored. More importantly, these cardiovascular diseases are associated with inflammation and infiltrated monocytes where BMP-7 has been demonstrated to be a key player in the differentiation of pro-inflammatory monocytes, or M1 macrophages, into anti-inflammatory M2 macrophages, which reduces developed cardiac dysfunction. Therefore, this review focuses on the molecular mechanisms of BMP-7 treatment in cardiovascular disease and its role as an anti-fibrotic, anti-apoptotic and anti-inflammatory growth factor, which emphasizes its potential therapeutic significance in heart diseases.

Evaluation of Anti-Inflammatory Properties of Herbal Aqueous Extracts and Their Chemical Characterization.

Plant extracts are gaining more attention as therapeutic agents against inflammation. In this study, four different widely used herbals were selected, such as holy basil leaf, sesame seed, long pepper, and cubeb pepper. We have evaluated the anti-inflammatory action of an aqueous extract from these herbs and tested their effects on monocyte-derived macrophages (MDMs). MDMs were pre-treated with these extracts individually for 2 h, followed by lipopolysaccharide (LPS) stimulation for 24 h and pro-inflammatory gene expression was analyzed. Also, we studied the effect of these extracts on the oxidation of low-density lipoprotein (LDL) by enzymatic (Myeloperoxidase) and non-enzymatic (copper) reactions. All extracts attenuated LPS-induced inflammation and also were able to inhibit the oxidation of LDL. These beneficial actions of extracts led us to identify molecules present in the extracts. A liquid chromatography-high resolution mass spectrometric analysis was performed to identify the chemical composition of extracts. Wide range of molecules were identified across all the extracts, short-chain organic acids, phenolic acids and derivatives, piperine and its structural homologues, eugenol, rosmarinic acid, flavonoids and their glucosides, and others. This study opens a door for future studies on non-pharmacological natural therapeutics that will be useful for consumers and producers, as well as industries utilizing bioactive compounds.

Alzheimer's Disease Markers in Aged ApoE-PON1 Deficient Mice.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging. Cardiovascular risk factors like hypertension and atherosclerosis increase the risk for AD. Polymorphic alleles of apolipoprotein E (ApoE) are one of the main genetic determinants of AD. OBJECTIVE: Mice, double-knockout (DKO) for ApoE (major cholesterol carrier in brain) and PON1 (paroxonase1, reduces oxidative stress), showed severe age-dependent atherosclerosis of the arteries carrying blood to the brain even on normal diet. This prompted us to investigate the possibility of an AD pathology resulting from the deficiency of ApoE and the induction of oxidative stress. METHODS: Atherosclerotic lesions were quantified by ImageJ. The brain hippocampus of young and old ApoE-PON1 DKO mice and control mice were harvested. RT-PCR analysis was performed for mRNA levels of AD specific markers. Blood levels of S100 calcium-binding protein B (S100B) protein were measured by ELISA. H&E as well as immunostaining was performed to detect amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) in brain tissues. Evans blue dye was used to evaluate the vascular permeability and blood-brain barrier (BBB) dysfunction. RESULTS: Results showed that the older DKO mice had severe carotid atherosclerosis, increased mRNA levels of AD markers in brain tissue, and elevated levels of serum S100B protein. Immunological staining confirmed the characteristics of AD. Ex-vivo imaging showed higher levels of Evans blue dye in the ApoE-PON1 DKO mice brain tissues, pointing toward impaired vasculature. CONCLUSION: Aged ApoE-PON1 DKO mice displaying AD specific markers along with Aß plaques, NFTs, and disrupted BBB suggests the animals are suffering from AD.

Primary prevention of atherosclerosis by pretreatment of low-density lipoprotein receptor knockout mice with sesame oil and its aqueous components.

Pharmacological intervention using statins and PCSK9 inhibitors have become first-line therapy in the prevention of hypercholesterolemia and atherosclerosis. Currently, no agent is available for the primary prevention of atherosclerosis. However, there is an emerging hypothesis that atherosclerosis could be driven by inflammation. In this study, we tested whether pretreatment with an aqueous extract from sesame oil (SOAE), which showed potent anti-inflammatory properties without hypocholesterolemic actions, would prevent subsequent atherosclerosis development in a mouse model. RAW 264.7 macrophages and female low-density lipoprotein receptor knockout (LDLR-/-) mice were used for in vitro and in vivo studies, respectively. Plasma lipids, cytokines and atherosclerotic lesions were quantified at the end of the study. RNA was extracted from the liver and aortic tissues and used for gene analysis. Pre-treatment of SOAE prevented Ox-LDL uptake by RAW macrophages and further inflammation in vitro. SOAE pre-treatment significantly reduced atherosclerotic lesions and pro-inflammatory gene expressions in LDLR-/- mice as compared to control mice. No significant change in plasma cholesterol levels was observed. A significant reduction in plasma levels of TNF-α, IL-6, MCP-1 and VCAM1 was observed in the SOAE pre-treated animals. This is the first study that demonstrates that pre-treatment with anti-inflammatory agents, could delay/decrease atherosclerosis.

Circulating platelet aggregates damage endothelial cells in culture.

BACKGROUND: Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). METHODS: Endothelial cells were grown in glass tubes and tagged with/without 0.5 µm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. RESULTS AND CONCLUSION: No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.

Nephro-protective action of P. santalinus against alcohol-induced biochemical alterations and oxidative damage in rats.

The present study investigated the antioxidant potential of P. santalinus heartwood methanolic extract (PSE) against alcohol-induced nephro-toxicity. The results indicated an increase in the concentration of kidney damage plasma markers, urea and creatinine with a concomitant decrease in the concentration of uric acid in alcohol-administered rats. A significant decrease in plasma electrolytes and mineral levels with increased kidney thiobarbituric acid reactive substances (TBARS) and nitric oxide (NOx) levels was also observed. PSE treatment to alcohol-administered rats effectively prevented the elevation in TBARS and NOx levels. Decreased activity of Na+/K+-ATPase in alcohol administered rats was brought to near normal levels with treatment of PSE. Chronic alcohol consumption affects antioxidant enzymatic activity and reabsorption function of the kidney which is evident from the decreased level of GSH as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione s-transferase (GST). However, treatment with PSE to alcohol-administered rats significantly enhanced these enzymatic activities and reduced glutathione (GSH) content close to normal level. Alcohol-induced organ damage was evident from morphological changes in the kidney. Nevertheless, administration of PSE effectively restored these morphological changes to normal. The flavonoid and tannoid compounds might have protective activity against alcohol-induced oxidative/nitrosative stress mediated kidney damage.

Increased presence of oxidized low-density lipoprotein in the left ventricular blood of subjects with cardiovascular disease.

OxidizedLDL(Ox-LDL) and oxidative stress have been implicated in both atherosclerosis and congestive heart failure (HF) development. Here, we tested whether Ox-LDLlevels in left ventricular blood (LVB) might differ from those of venous peripheral blood (PB), and whether the level might depend on cardiac function. We also tested whether theLDLmolecule is likely to have a longer residence time in the left ventricle ofHFsubjects with low ejection fraction (EF). The aim of this study was to determine Ox-LDLlevels, paraoxonase 1 (PON1) activity, and cholesterol efflux capacity (CEC) ofPBandLVB, and correlate these values withLVEF Sixty-oneHFpatients underwent preoperative transthoracic echocardiographic assessment of ventricular function.LVEFs were determined using Simpson's biplane technique.LVBandPBlevels of Ox-LDLwere determined, andPON1 activity and plasma cholesterol efflux capacity were measured. A significant increase in the levels of Ox-LDLinLVBwas noted as compared to levels inPB, even whenEFwas near normal. However, as ejection fraction decreased, the level of Ox-LDLinPBapproached that of theLVBPON1 activity and cholesterol efflux studies indicated increased oxidative stress inLVBand a decreased ability to promote cholesterol efflux from lipid-enriched macrophages. The results suggest thatLVBis more oxidatively stressed compared toPB, and thereforeLVtissue might be affected differently than peripheral tissues. We recently reported that brain natriuretic peptide (BNP), a marker forHF, is induced by Ox-LDL, so it is possible localized factors within theLVcould profoundly affect markers ofHF.

Atherosclerosis--do we know enough already to prevent it?

In this review, we have briefly summarized the characteristics of lipids and lipoproteins and the atherosclerotic process. The development of atherosclerosis is a continuous process that involves numerous cellular and acellular processes that influence the behavior of each other. These include oxidative stress, lipoprotein modifications, macrophage polarization, macrophage lipid accumulation, generation of pro- and anti-inflammatory components, calcification, cellular growth and proliferation, and plaque rupture. The precise role(s) of many of these are unknown. Understanding the events at each particular stage might shed more light onto the process as a whole and could potentially reveal targets for intervention. Therapeutic modalities that work at one stage may have little to no influence on other stages of the disease.

Aspirin may influence cellular energy status.

In our previous findings, we have demonstrated that aspirin/acetyl salicylic acid (ASA) might induce sirtuins via aryl hydrocarbon receptor (Ah receptor). Induction effects included an increase in cellular paraoxonase 1 (PON1) activity and apolipoprotein A1 (ApoA1) gene expression. As predicted, ASA and salicylic acid (SA) treatment resulted in generation of H2O2, which is known to be an inducer of mitochondrial gene Sirt4 and other downstream target genes of Sirt1. Our current mass spectroscopic studies further confirm the metabolism of the drugs ASA and SA. Our studies show that HepG2 cells readily converted ASA to SA, which was then metabolized to 2,3-DHBA. HepG2 cells transfected with aryl hydrocarbon receptor siRNA upon treatment with SA showed the absence of a DHBA peak as measured by LC-MS/MS. MS studies for Sirt1 action also showed a peak at 180.9 m/z for the deacetylated and chlorinated product formed from N-acetyl lε-lysine. Thus an increase in Sirt4, Nrf2, Tfam, UCP1, eNOS, HO1 and STAT3 genes could profoundly affect mitochondrial function, cholesterol homeostasis, and fatty acid oxidation, suggesting that ASA could be beneficial beyond simply its ability to inhibit cyclooxygenase.

Cationic peptides neutralize Ox-LDL, prevent its uptake by macrophages, and attenuate inflammatory response.

OBJECTIVE: Apolipoprotein A1 (ApoA1) and apolipoprotein E (ApoE) mimetic peptides have attracted attention due to their ability to reduce atherosclerosis and exhibit antioxidant, anti-inflammatory, and hypolipidemic properties. In this study, we tested whether three distinct and unrelated cationic peptides would inhibit the oxidation of lipoproteins and whether they would counteract and neutralize the negatively charged modified lipoproteins, inhibit their uptake and inflammation by macrophages. METHODS AND RESULTS: 5F-mimetic peptide of ApoA1, LL27 derived from the anti-microbial peptide hCAP, and a human glycodelin derived peptide were commercially synthesized. We noted that these three distinct cationic lysine-rich peptides, two of which were unrelated to any known apolipoproteins, inhibited copper-mediated oxidation of lipoproteins and reduced lipid peroxides in a lysine dependent manner. The peptides also retarded the electrophoretic mobility of previously oxidized LDL and acetylated LDL by virtue of their net positive charge. Pre-incubation of peptides with modified lipoproteins reduced the uptake of the latter by macrophages, thus preventing the formation of foam cells. The cationic peptides inhibited oxidized LDL (Ox-LDL)-induced inflammatory response both in vitro and in vivo. CONCLUSION: Based on these results, we suggest that in addition to the well known mimetic peptides, other suitable cationic peptides may be of use for controlling Ox-LDL mediated inflammation and atherosclerotic progression.

Aspirin may promote mitochondrial biogenesis via the production of hydrogen peroxide and the induction of Sirtuin1/PGC-1α genes.

Based on the rapid hydrolysis of acetyl salicylic acid (ASA, Aspirin) to salicylic acid (SA), the ability of SA to form dihydroxy benzoic acid (DBA), and the latter's redox reactions to yield hydrogen peroxide (H(2)O(2)), we predicted that ASA may have the potential to induce Sirtuin1 (Sirt1) and its downstream effects. We observed that treatment of cultured liver cells with ASA resulted in the induction of Sirt1, peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α), and NAD(P)H quinone oxidoreductase 1 (Nqo1) genes. Paraoxonase 1 (PON1) and Aryl hydrocarbon receptor (AhR) siRNA transfections inhibited the induction of gene expressions by ASA suggesting the need for the acetyl ester hydrolysis and hydroxylation to DHBA. The latter also induced Sirt1, confirming the proposed pathway. As predicted, ASA and SA treatment resulted in the production of H(2)O(2), a known inducer of Sirt1 and confirmed in the current studies. More importantly, ASA treatment resulted in an increase in mitochondria as seen by tracking dyes. We suggest that DHBA, generated from ASA, via its oxidation/reduction reactions mediated by Nqo1 might be involved in the production of O(2)(-.) and H(2)O(2). As Sirt1 and PGC-1α profoundly affect mitochondrial metabolism and energy utilization, ASA may have therapeutic potential beyond its ability to inhibit cyclooxygenases.

Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages.

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1ß, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1ß, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.