Vascular Protector Research Articles

Performance of nitric oxide in sepsis: a scoping review

Performance of nitric oxide in sepsis: a scoping review

Prosopis alba seed flour improves vascular function in a rabbit model of high fat diet-induced metabolic syndrome

AimsProsopis alba flour is a natural source of nutrient and phytochemicals with potential effects on cardiovascular risk factors. The aim of this work was to examine the effects of dietary supplementation with Prosopis alba seed flour (Pr-Feed) on a high fat diet (FD)-induced rabbit model of metabolic syndrome. Main methodsRabbits were separated in four groups: fed regular diet (CD); CD supplemented with Pr-Feed; fed on 18 % FD; FD supplemented with Pr-Feed. All diets were administrated for 6 weeks. After the feeding period body weights, mean blood pressure, heart rate and visceral abdominal fat (VAF) were determined; glucose tolerance test (GTT) was performed; total cholesterol (TC), HDL-cholesterol, LDL-cholesterol, triglycerides (TG), fasting glucose (FG), aspartate amino transferase, alanine amino transferase, bilirubin and creatinine were measured in serum. Abdominal aorta was excised and vascular function was assessed by acetylcholine relaxation and contractile response to KCl, norepinephrine and angiotensin II. Key findingsPhytochemical analyses showed that the main compounds of Pr-Feed were apigenin C-glycosides. FD increased VAF, FG, TG, reduced HDL-cholesterol and induced abnormal GTT. Pr-Feed addition to FD did not modify these alterations. Aortic rings from rabbits fed on FD exhibited an impaired relaxation-response to acetylcholine and increased agonist vasoconstrictor responses. Pr Feed-supplemented FD improved the response to acetylcholine, and prevented the increase of the contractile response to KCl, norepinephrine and angiotensin II. SignificanceResults suggest that dietary supplementation with Pr-Feed, rich in apigenin C-glycosides, has vascular protector properties and could be used to prevent vascular alterations characterizing the metabolic syndrome.

Creating a mouse model resistant to induced ischemic stroke and cardiovascular damage

Vascular prostanoids, isomerized from an intermediate prostaglandin (PG), H2, produced by cyclooxygenase (COX), exert various effects on the vascular system, both protective and destructive. During endothelial dysfunction, vascular protector prostacyclin/prostaglandin I2 (PGI2) is decreased, while inflammatory PGE2 and thrombotic TXA2 are increased. Therefore, our research aim was to reverse the event by controlling PGH2 metabolism by generating an in vivo model via enzymatic engineering of COX-1 and prostacyclin synthase (PGIS). The COX-1 and PGIS genes were linked to a 10-residue amino acid linker to form a Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGIS. Transgenic (CP-Tg) mice in a FVB/N background were generated using the pronuclear microinjection method. We first confirmed mRNA and protein expression of COX-1-10aa-PGIS in various CP-Tg mouse tissues, as well as upregulation of circulating PGI2. We then examined the cardiovascular function of these mice. Our CP-Tg mice exhibited marked resistance to vascular assault through induced carotid arterial blockage, acute thrombotic stroke and arterial arrest, angiotensin-induced peripheral vasoconstriction, and hepatic lipid accumulation after receiving a high-fat diet. They also had a longer lifespan compared with wild-type mice. This study raises the possibility of fighting cardiovascular diseases by regulating cellular arachidonic acid-derived PGH2 metabolites using enzymatic engineering.

Interleukin-10: a pleiotropic regulator in pregnancy.

Pregnancy is a unique and well-choreographed physiological process that involves intricate interplay of inflammatory and anti-inflammatory milieu, hormonal changes, and cellular and molecular events at the maternal-fetal interface. IL-10 is a pregnancy compatible cytokine that plays a vital role in maintaining immune tolerance. A wide array of cell types including both immune and non-immune cells secret IL-10 in an autocrine and paracrine manner. IL-10 binds to a specific receptor complex and activates JAK-STAT and PI3K-Akt signaling pathways while inhibiting NF-κB signaling pathway. IL-10 exerts its anti-inflammatory effects mainly by decreasing pro-inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF-α, by inducing heme oxygenase-1, and by inhibiting antigen presentation via blocking major histocompatibility complex (MHC) class II expression. Prior studies from our group and others have shown that IL-10 also functions as a potent protector against vascular dysfunction, and enhancement of IL-10 may serve as an immunotherapeutic intervention to treat adverse pregnancy outcomes. This review seeks to critically evaluate the archetypal functions of IL-10 as an immune suppressive factor as well as its novel functions as a vascular protector and modulator of endoplasmic reticulum (ER) stress and autophagy in the context of normal and adverse pregnancy outcomes.

Establishing novel prostacyclin-synthesizing cells with therapeutic potential against heart diseases

BackgroundFor decades, there have been many ongoing attempts to use prostaglandin I2 (PGI2) to treat heart diseases, such as pulmonary arterial hypertension. However, the short half life of PGI2 has limited the therapeutic impact potential. MethodsHere, we have engineered a novel adipose tissue-derived cell that constantly produces PGI2, through transfecting of an engineered cDNA of a hybrid enzyme (human COX-1-10-aa-PGIS) which has superior triple catalytic functions in directly converting arachidonic acid into PGI2. ResultsThe gene-transfected cells were further converted into a stable cell line, in which cells constantly express the hybrid enzyme and are capable of producing large-amounts of PGI2. In a comparison between un-transfected- and gene-transfected cells, it was determined that the majority of the endogenous AA metabolism shifted from that of unwanted PGE2 (in un-transfected cells) to that of the preferred PGI2 (in gene-transfected cells) with a PGI2/PGE2 ratio change from 0.03 to 25. The PGI2-producing cell line not only exhibited an approximate 50-fold increase in PGI2 biosynthesis, but also demonstrated superior anti-platelet aggregation in vitro, and increased reperfusion in the mouse ischemic hindlimb model in vivo. ConclusionsThe cells, which have an ability to increase the biosynthesis of the vascular protector, PGI2, while reducing that of the vascular inflammatory mediator, PGE2, provide a dual effect on vascular protection, which is not available through any existing drug treatments. Thus, the current finding has potential to be an experimental intervention for PGI2-deficient heart diseases, such as pulmonary arterial hypertension.

Novel Mechanism of the Vascular Protector Prostacyclin: Regulating MicroRNA Expression

Prostacyclin (PGI(2)) is a key vascular protector, metabolized from endogenous arachidonic acid (AA). Its actions are mediated through the PGI(2) receptor (IP) and nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ). Here, we found that PGI(2) is involved in regulating cellular microRNA (miRNA) expression through its receptors in a mouse adipose tissue-derived primary culture cell line expressing a novel hybrid enzyme gene (COX-1-10aa-PGIS), cyclooxygenase-1 (COX-1) and PGI(2) synthase (PGIS) linked with a 10-amino acid linker. The triple catalytic functions of the hybrid enzyme in these cells successfully redirected the endogenous AA metabolism toward a stable and dominant production of PGI(2). The miRNA microarray analysis of the cell line with upregulated PGI(2) revealed a significant upregulation (711, 148b, and 744) and downregulation of miRNAs of interest, which were reversed by antagonists of the IP and PPARγ receptors. Furthermore, we also found that the insulin-mediated lipid deposition was inhibited in the PGI(2)-upregulated adipocytes. The study also initiated a discussion that suggested that the endogenous PGI(2) inhibition of lipid deposition in adipocytes could involve miRNA-mediated inhibition of expression of the targeted genes. This indicated that PGI(2)-miRNA regulation could exist in broad pathophysiological processes involving PGI(2) (i.e., apoptosis, vascular inflammation, cancer, embryo implantation, and obesity).

Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: Mechanisms of COX-2 inhibitor risk to heart disease

AimOur aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher “priority” to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs. Main methodsCOX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET. Key findingsThe Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0μM; however, it was 3-fold more (~6.0μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0μM and 20s, respectively, which were 2–5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS–CFP is shorter than that between COX-1-RFP and PGIS–CFP. SignificanceThe study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.

Generation of Transgenic Mice Overexpressing a Hybrid Enzyme Up‐regulating Biosynthesis of Vascular Protector Prostacyclin

Prostacyclin (PGI2) is a potent vascular protector through its anti‐platelet aggregation and vasodilation properties. Non‐steroid anti‐inflammatory drugs (NSAIDs) by inhibiting PGI2 production promote heart diseases. Recently, we have successfully engineered a hybrid enzyme (COX‐1‐10aa‐PGIS, by linking cyclooxygenase isoform 1(COX‐1) with PGI2 synthase (PGIS) through a 10 amino acids (10aa) linker) with triple catalytic functions directly converting arachidonic acid into PGI2. PGI2 biosynthesis was upregulated in the mammalian cells, transfected with the hybrid enzyme cDNA. Thus, with the objective of translating these in vitro results into in vivo, we generated transgenic mice overexpressing COX‐1‐10aa‐PGIS. PCR using mouse tail tissue confirmed the incorporation of COX‐1‐10aa‐PGIS gene in the transgenic mouse chromosome. Permanent expression of the hybrid enzyme was identified by Western blot. The catalytic activity of the hybrid enzyme to form PGI2 was determined by enzyme activity assay and LC/MS analysis of transgenic mouse tail and urine, respectively. In conclusion, a transgenic mouse overexpressing the hybrid enzyme with upregulated PGI2 biosynthesis was successfully obtained. The mice can be used to further uncover the mechanisms of the vascular protection of PGI2 and test the side effects of NSAIDs.Funding: R01 HL56712 (for KHR); RO1HL079389 (for KHR) and RC1 HL100807 (for RD and KHR)

Endogenous Prostacyclin Signaling Regulating MicroRNA Expression in Mammalian Cells

Prostacyclin (PGI2), a key vascular protector and a representative of eicosanoids metabolized from endogenous arachidonic acid (AA), involved in regulating cellular microRNA expression was found. A primary cultured cell line derived from mouse adipose tissue containing the pathway enzymes to metabolize endogenous AA to diverse eicosanoids was transfected with a cDNA of an engineered hybrid synthases which integrated cyclooxygenase‐1 and PGI2 synthase (PGIS) triple catalytic functions. It successfully redirected the cells (PGI2‐cells) stably and dominantly metabolizing the endogenous AA to PGI2. By microarray analysis for 119 miRNAs, expression of miRNA‐711 and 466f‐3p in the PGI2‐cells was dramatically (approximately 15‐folds) upregulated and down regulated, respectively. The up‐ and down‐ regulated miRNAs expression in the PGI2‐cells were completely abolished by specifically inhibiting either the PGI2 signalings through the membrane receptor or the PPARγ pathway. This study provides the evidences that PGI2 could be involved in much broad pathophysiological processes including the controlling of apoptosis, vascular inflammation, atherosclerosis, cancer, implantation, and obesity through its action on up‐ and down‐regulation of the miRNA expressions. The study has created a new dimension to understand the diverse biological functions of eicosanoids derived from endogenous AA in general.

An active triple‐catalytic hybrid enzyme engineered by linking cyclo‐oxygenase isoform‐1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003-14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI(2)) synthase (PGIS) for the direct conversion of arachidonic acid into PGI(2) through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI(2), in cells is an ideal model for the prevention and treatment of thromboxane A(2) (TXA(2))-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI(2) synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3-5-fold higher expression capacity and better PGI(2)-synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI(2) was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI(2), revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI(2) production while decreasing TXA(2) production) in TXA(2) synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI(2) biosynthesis in a human cell line, which provides a basis for developing a PGI(2)-producing therapeutic cell line for use against vascular diseases.

Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential

Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI 2) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI 2 [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003–14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield (∼4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells’ membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions ( K m = 3 μM and K cat = 100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI 2 than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 μg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI 2, and revealed its potential for anti-thrombosis therapeutics.

Increased serum adiponectin by heme oxygenase‐1 in obese diabetic zucker rats increases insulin sensitivity and glucose tolerance

We hypothesized that heme oxygenase‐1 (HO‐1) attenuates diabetic complications via stimulation of a potent vascular protector including adiponectin in rodent with type 2 diabetes. We examined the effect of cobalt protoporphyrin (CoPP) on serum levels of adiponectin in obese and obsess‐diabetic Zucker rats. The onset of diabetes or obesity coincided with a decrease in HO activity, which was restored by administration of CoPP. Upregulation of HO‐1 expression in diabetic rats produced an increase in adiponectin (p<0.003), decreased superoxide (p<0.05) and reduced blood pressure (p<0.01). Increased HO activity was associated with an increase in insulin sensitivity, glucose tolerance and significant reduction in body weight and fat content. The increases in HO‐1‐adiponectin resulted in a decrease within TNF, IL‐1 and IL‐6. The effect of HO‐1 on human MSC show similar results, increases in HO‐1 and decreased adipogenesis. The administration of an inhibitor of HO activity decreased adiponectin. This study suggests that the anti‐diabetic/anti‐obesity effect of HO‐1 is due to regulation of adipogenesis, increases adiponectin secretion, decreases TNF, IL‐1 and IL‐6. HO‐1 treatment was associated with the restoration of insulin sensitivity, preserve pancreatic insulin response to glucose, reduction in weight gains and the amelioration vascular diseases. This work is supported by NIH grants HL55601 and HL34300.

Engineering of a Protein with Cyclooxygenase and Prostacyclin Synthase Activities That Converts Arachidonic Acid to Prostacyclin

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.