Chick Embryo Ex Vivo Aortic Sprouting Assays for Cardiovascular Research.

Angiogenesis is the formation of new blood vesicles and is controlled by a dynamic cascade of molecular and cellular activities. The whole procedure can be replicated in vitro under chemically specified conditions by cultivating chick aortic explants in biomatrices. In this technique, angiogenesis is powered by endogenous molecules that the aorta releases to promote its outgrowth. In an ordered series of morphogenetic events, sprouting endothelial cells are strongly associated with macrophages, fibroblasts, and pericytes, recapitulating all the phases of the angiogenic process. The structural, morphologic, and molecular properties of the angiogenic process can be studied and the effectiveness of pro/antiangiogenic drugs can also be evaluated using this aortic culture. We describe in this chapter the basic procedure currently used in our laboratory to measure the angiogenic properties for cardiovascular research.

Flavonoids: Classification, Function, and Molecular Mechanisms Involved in Bone Remodelling.

Flavonoids are polyphenolic compounds spotted in various fruits, vegetables, barks, tea plants, and stems and many more natural commodities. They have a multitude of applications through their anti-inflammatory, anti-oxidative, anti-carcinogenic properties, along with the ability to assist in the stimulation of bone formation. Bone, a rigid connective body tissue made up of cells embedded in a mineralised matrix is maintained by an assemblage of pathways assisting osteoblastogenesis and osteoclastogenesis. These have a significant impact on a plethora of bone diseases. The homeostasis between osteoblast and osteoclast formation decides the integrity and structure of the bone. The flavonoids discussed here are quercetin, kaempferol, icariin, myricetin, naringin, daidzein, luteolin, genistein, hesperidin, apigenin and several other flavonoids. The effects these flavonoids have on the mitogen activated protein kinase (MAPK), nuclear factor kappa ß (NF-kß), Wnt/ß-catenin and bone morphogenetic protein 2/SMAD (BMP2/SMAD) signalling pathways, and apoptotic pathways lead to impacts on bone remodelling. In addition, these polyphenols regulate angiogenesis, decrease the levels of inflammatory cytokines and play a crucial role in scavenging reactive oxygen species (ROS). Considering these important effects of flavonoids, they may be regarded as a promising agent in treating bone-related ailments in the future.

Evaluation of the angiogenic properties of Brugia malayi asparaginyl-tRNA synthetase and its mutants: A study on the molecular target for antifilarial drug development.

Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS) has been identified as an immunodominant antigen and a physiocrine that mimics Interleukin-8 (IL-8) to induce chemotaxis and angiogenesis in endothelial cells. Computational analyses have shown that the N-terminal region of BmAsnRS has a novel fold, a lysine rich ß-hairpin α-helix, (FLIRTKKDGKQIWE) which is similar to that present in IL-8 chemokine, CXCR1. This novel fold is involved in tRNA binding and is integral for the manifestation of the disease, lymphatic filariasis (LF). Drug discovery programmes carried out so far for LF have not been successful because of the target (BmAsnRS) resistance due to the disease-associated mutation. Mutations in AARS targets have been shown to correlate with several diseases. However, no disease-associated mutational studies have been carried out for LF. BmAsnRS has been an established target for LF. It was proposed, therefore, to study the effect of single point mutations in BmAsnRS so as to elucidate the molecular target. An understanding of the molecular consequences of mutations will provide insight into how resistance develops in addition to the identification of the likely resistance-conferring mutations. Three mutants were, therefore, generated by site-directed mutagenesis using CUPSAT server and their angiogenic properties evaluated. Cytometric analysis of the mutants on endothelial cell cycle was also carried out. CUPSAT prediction of protein stability upon point mutations reveal that two mutants generated are likely resistance-conferring mutations. All the three mutants show significant reduction in their angiogenic properties and reduction in the DNA content in the cells of S and G2/M phases thus showing altered function of the gene encoding the drug target. The resistance- conferring mutants, however, show angiogenic properties nearer to the wild type protein, BmAsnRS. Future work on designing newer drugs may take into consideration these drug resistance-conferring mutations.

LncRNA MALAT1 Promotes Tumor Angiogenesis by Regulating MicroRNA-150-5p/VEGFA Signaling in Osteosarcoma: In-Vitro and In-Vivo Analyses.

The present study aims to analyze the expression of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in human osteosarcoma (OS) cells and to investigate its role in OS-induced angiogenesis. MALAT1 expression in OS cells was significantly higher than in normal osteoblasts. The functional analysis indicated that MALAT1 appears to enhance OS-induced angiogenesis, in vitro and in vivo analyses, endothelial cell proliferation and migration, chick embryo angiogenesis assay, and zebrafish xenograft model. Mechanistically, silencing MALAT1 downregulated vascular endothelial growth factor A (VEGFA) expression and upregulated miR-150-5p expression in OS cells, and MALAT1-mediated angiogenic induction by VEGFA in OS microenvironment. Moreover, MALAT1 directly targeted miR-150-5p and miR-150-5p directly target VEGFA in OS. Overexpression of miR-150-5p downregulates VEGFA expression in OS. More notably, we showed that MALAT1 induced angiogenesis in OS microenvironment by upregulating the expression of VEGFA via targeting miR-150-5p. Overall, our findings suggest that MALAT1 promotes angiogenesis by regulating the miR-150-5p/VEGFA signaling in OS microenvironment. The findings of the molecular mechanisms of MALAT1 in tumor angiogenesis offer a new viewpoint on OS treatment.

Recent Insight on the Non-coding RNAs in Mesenchymal Stem Cell-Derived Exosomes: Regulatory and Therapeutic Role in Regenerative Medicine and Tissue Engineering.

Advances in the field of regenerative medicine and tissue engineering over the past few decades have paved the path for cell-free therapy. Numerous stem cell types, including mesenchymal stem cells (MSCs), have been reported to impart therapeutic effects via paracrine secretion of exosomes. The underlying factors and the associated mechanisms contributing to these MSC-derived exosomes' protective effects are, however, poorly understood, limiting their application in the clinic. The exosomes exhibit a diversified repertoire of functional non-coding RNAs (ncRNAs) and have the potential to transfer these biologically active transcripts to the recipient cells, where they are found to modulate a diverse array of functions. Altered expression of the ncRNAs in the exosomes has been linked with the regenerative potential and development of various diseases, including cardiac, neurological, skeletal, and cancer. Also, modulating the expression of ncRNAs in these exosomes has been found to improve their therapeutic impact. Moreover, many of these ncRNAs are expressed explicitly in the MSC-derived exosomes, making them ideal candidates for regenerative medicine, including tissue engineering research. In this review, we detail the recent advances in regenerative medicine and summarize the evidence supporting the altered expression of the ncRNA repertoire specific to MSCs under different degenerative diseases. We also discuss the therapeutic role of these ncRNA for the prevention of these various degenerative diseases and their future in translational medicine.

Application of Artificial Neural Network as a nonhazardous alternative on kinetic analysis and modeling for green synthesis of cobalt nanocatalyst from Ocimum tenuiflorum.

The present paper is dedicated to analyze non-hazardous kinetic behaviour and modelling of green synthesized cobalt nanocatalyst (CoNCs), using an Artificial Neural Network (ANN). In order to supplement the trace metal in other applications, CoNCs were rapidly synthesized with a Cobalt sulphate solution at room temperature between 30 and 35 ºC at pH 7.2 under less reaction time. The Levenberg - Marquardt algorithm (LM) is used to investigate the experimental values by applying ANN. The results of variance using logistic ANN model depicts that the maximum nanoparticles were synthesized at its optimized stipulation of 0.5 h stirring time, 25 mL volume of extract and 20 mL volume of cobalt sulphate. The developed ANN model proved to be an efficient size determining tool in the biosynthesis of cobalt nanocatalyst. Experimental behavior using potentiometric analysis confirms that the linearity in CoNCs formation and size coincides (5-38 nm)with the predicted values of the ANN model. Techno economic analysis proved that, green synthesis reduced 30-40% in raw material cost and 60% in energy consumption.

Identification and analysis of circulating long non-coding RNAs with high significance in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) lacks diagnostic biomarkers. Circulating long non-coding RNAs (lncRNAs) can serve as valuable diagnostic biomarkers in cardiovascular disease. To seek potential lncRNAs as a diagnostic biomarker for DCM, we investigated the genome-wide expression profiling of circulating lncRNAs and mRNAs in type 2 diabetic db/db mice with and without DCM and performed bioinformatic analyses of the deregulated lncRNA-mRNA co-expression network. Db/db mice had obesity and hyperglycemia with normal cardiac function at 6 weeks of age (diabetes without DCM) but with an impaired cardiac function at 20 weeks of age (DCM) on an isolated Langendorff apparatus. Compared with the age-matched controls, 152 circulating lncRNAs, 127 mRNAs and 3355 lncRNAs, 2580 mRNAs were deregulated in db/db mice without and with DCM, respectively. The lncRNA-mRNA co-expression network analysis showed that five deregulated lncRNAs, XLOC015617, AK035192, Gm10435, TCR-α chain, and MouselincRNA0135, have the maximum connections with differentially expressed mRNAs. Bioinformatic analysis revealed that these five lncRNAs were highly associated with the development and motion of myofilaments, regulation of inflammatory and immune responses, and apoptosis. This finding was validated by the ultrastructural examination of myocardial samples from the db/db mice with DCM using electron microscopy and changes in the expression of myocardial tumor necrosis factor-α and phosphorylated p38 mitogen-activated protein kinase in db/db mice with DCM. These results indicate that XLOC015617, AK035192, Gm10435, TCR-α chain, and MouselincRNA0135 are crucial circulating lncRNAs in the pathogenesis of DCM. These five circulating lncRNAs may have high potential as a diagnostic biomarker for DCM.

Assessment of human ovarian follicular fluid derived mesenchymal stem cells in chitosan/PCL/Zn scaffold for bone tissue regeneration.

Bone tissue engineering compasses the use of mesenchymal stem cells (MSCs) along with engineered biomaterial construct to augment bone regeneration. Till now, MSCs were isolated from various sources and used in cellular constructs. For the first time, in this study, MSCs were isolated from human Ovarian Follicular Fluid (OFF) and characterized by CD 44+ and CD 105+ markers via confocal microscopy and flow cytometry. Additionally, MSCs stemness, proliferation and colony-forming unit ability, multi-lineage differentiation potential were also studied. To test its suitability for bone tissue engineering applications, we grew the MSCs with the conditioned medium obtained from biocomposite scaffold by fusing a natural polymer, Chitosan (CS) and a synthetic polymer, Polycaprolactone (PCL) and the scaffold were coated with Zinc divalent ions to impart osteogenic properties. The physico-chemical characterization of scaffold, such as FTIR, XRD, and SEM studies was carried out. The biological characterization showed that the scaffolds were compatible with MSCs and promoted osteoblast differentiation which was confirmed at both cellular and molecular levels. The cellular construct increased calcium deposition, analyzed by alizarin red staining and ALP activity at cellular level. At the molecular level, the osteoblast markers expression such as Runx2 and type 1 collagen mRNAs, and osteonectin (ON) and osteocalcin (OC) secretory proteins were increased in the presence of scaffold. Overall, the current study recommends that MSCs can be easily obtained from human waste OFF, and grown in standard in vitro conditions. Successful growth of such MSCs with CS/PCL/Zn scaffold opens new avenues in utilizing the cell source for bone tissue engineering.

Feasibility of biodiesel production from waste cooking oil: lab-scale to pilot-scale analysis.

In the last few decades, consciousness of fossil fuel resources and increased environmental concerns have given the need for emergence of alternative fuel. Biodiesel is one of the potential renewable energies produced from edible and non-edible biomass which could be a potential alternative for petrol-derived diesel. In this work, initially the process of biodiesel production from waste cooking oil using potassium hydroxide as catalyst and the process parameters were studied in laboratory. The maximum biodiesel yield of 97% was attained at 75 °C with 1 wt% catalyst concentration and oil-methanol molar ratio of 1:06 at 350 rpm and 90 min. Also, these process conditions were used for biodiesel production in the pilot plant and obtained 97% yield. Overall, mass balance for the pilot plant was studied to analyze the product yield loss. The fatty acid methyl ester formation in the plant was confirmed by characterization with FTIR and 1H NMR. Further, the quality of biodiesel produced was compared for its physiochemical properties with the ASTM standards.

Current status and strategies of long noncoding RNA research for diabetic cardiomyopathy.

Long noncoding RNAs (lncRNAs) are endogenous RNA transcripts longer than 200 nucleotides which regulate epigenetically the expression of genes but do not have protein-coding potential. They are emerging as potential key regulators of diabetes mellitus and a variety of cardiovascular diseases. Diabetic cardiomyopathy (DCM) refers to diabetes mellitus-elicited structural and functional abnormalities of the myocardium, beyond that caused by ischemia or hypertension. The purpose of this review was to summarize current status of lncRNA research for DCM and discuss the challenges and possible strategies of lncRNA research for DCM. A systemic search was performed using PubMed and Google Scholar databases. Major conference proceedings of diabetes mellitus and cardiovascular disease occurring between January, 2014 to August, 2018 were also searched to identify unpublished studies that may be potentially eligible. The pathogenesis of DCM involves elevated oxidative stress, myocardial inflammation, apoptosis, and autophagy due to metabolic disturbances. Thousands of lncRNAs are aberrantly regulated in DCM. Manipulating the expression of specific lncRNAs, such as H19, metastasis-associated lung adenocarcinoma transcript 1, and myocardial infarction-associated transcript, with genetic approaches regulates potently oxidative stress, myocardial inflammation, apoptosis, and autophagy and ameliorates DCM in experimental animals. The detail data regarding the regulation and function of individual lncRNAs in DCM are limited. However, lncRNAs have been considered as potential diagnostic and therapeutic targets for DCM. Overexpression of protective lncRNAs and knockdown of detrimental lncRNAs in the heart are crucial for defining the role and function of lncRNAs of interest in DCM, however, they are technically challenging due to the length, short life, and location of lncRNAs. Gene delivery vectors can provide exogenous sources of cardioprotective lncRNAs to ameliorate DCM, and CRISPR-Cas9 genome editing technology may be used to knockdown specific lncRNAs in DCM. In summary, current data indicate that LncRNAs are a vital regulator of DCM and act as the promising diagnostic and therapeutic targets for DCM.

Uncoupling Warburg effect and stemness in CD133+ve cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia.

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca2+ signaling and cell cycle analysis. CD133+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133+ve cells. In summary, both CD133+ve/-ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of ß-catenin.

BACKGROUND: Cardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation. METHODS: CDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot. RESULTS: Results revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, ß-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho ß-catenin while non phospho ß-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs. CONCLUSION: Combined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.

Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action.

Vicious cycles of mutations and reactive oxygen species (ROS) generation contribute to cancer progression. The use of antioxidants to inhibit ROS generation promotes cytostasis by affecting the mutation cycle and ROS-dependent survival signaling. However, cancer cells select mutations to elevate ROS albeit maintaining mitochondrial hyperpolarization (Δψm), even under hypoxia. From this perspective, the use of drugs that disrupt both ROS generation and Δψm is a viable anticancer strategy. Hence, we studied the effects of mitochondrially targeted carboxy proxyl nitroxide (Mito-CP) and a control ten carbon TPP moiety (Dec-TPP+) in the human Burkitt lymphoma cell line (Daudi) and normal peripheral blood mononuclear cells under hypoxia and normoxia. We found preferential localization, Δψm and adenosine triphosphate loss, and significant cytotoxicity by Mito-CP in Daudi cells alone. Interestingly, ROS levels were decreased and maintained in hypoxic and normoxic cancer cells, respectively, by Mito-CP but not Dec-TPP+, therefore preventing any adaptive signaling. Moreover, dual effects on mitochondrial bioenergetics and ROS by Mito-CP curtailed the cancer survival via Akt inhibition, AMPK-HIF-1α activation and promoted apoptosis via increased BCL2-associated X protein and poly (ADP-ribose) polymerase expression. This dual mode of action by Mito-CP provides a better explanation of the application of antioxidants with specific relevance to cancerous transformation and adaptations in the Daudi cell line.

MicroRNA: A new therapeutic strategy for cardiovascular diseases.

Myocardial infarction, atherosclerosis, and hypertension are the most common heart-related diseases that affect both the heart and the blood vessels. Multiple independent risk factors have been shown to be responsible for cardiovascular diseases. The combination of a healthy diet, exercise, and smoking cessation keeps these risk factors in check and helps maintain homeostasis. The dynamic monolayer endothelial cell integrity and cell-cell communication are the fundamental mechanisms in maintaining homeostasis. Recently, it has been revealed that small noncoding RNAs (ncRNAs) play a critical role in regulation of genes involved in either posttranscriptional or pretranslational modifications. They also control diverse biological functions like development, differentiation, growth, and metabolism. Among ncRNAs, the short interfering RNAs (siRNAs), and microRNAs (miRNAs) have been extensively studied, but their specific functions remain largely unknown. In recent years, miRNAs are efficiently studied as one of the important candidates for involvement in most biological processes and have been implicated in many human diseases. Thus, the identification and the respective targets of miRNAs may provide novel molecular insight and new therapeutic strategies to treat diseases. This review summarizes the recent developments and insight on the role of miRNAs in cardiovascular disease prognosis, diagnostic and clinical applications.

Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis.

Thrombopoietin receptor agonists protect human cardiac myocytes from injury by activation of cell survival pathways.

Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart. Eltrombopag is a small molecule agonist of the thrombopoietin receptor, the physiologic target of thrombopoietin. However, the ability of eltrombopag and thrombopoietin to protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (n = 6-10/group) were treated with eltrombopag (0.1-30.0 µM) or thrombopoietin (0.1-30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N2/5% CO2) and 16 hours of reoxygenation to determine their ability to confer resistance to myocardial injury. The thrombopoietin receptor c-Mpl was detected in unstimulated human cardiac myocytes by Western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner, with an optimal concentration of 3 µM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple prosurvival pathways; inhibition of Janus kinase-2, proto-oncogene tyrosine-protein kinase, protein kinase B/phosphatidylinositol-3 kinase, p44/42 mitogen-activated protein kinase (MAPK), and p38 MAPK abolished cardiac myocyte protection by eltrombopag and thrombopoietin. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer a long-lasting benefit through activation of prosurvival pathways during ischemia.

20-HETE increases survival and decreases apoptosis in pulmonary arteries and pulmonary artery endothelial cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P-450 product present in vascular smooth muscle and uniquely located in the vascular endothelium of pulmonary arteries (PAs). 20-HETE enhances reactive oxygen species (ROS) production of bovine PA endothelial cells (BPAECs) in an NADPH oxidase-dependent manner and is postulated to promote angiogenesis via activation of this pathway in systemic vascular beds. We tested the capacity of 20-HETE or a stable analog of this compound, 20-hydroxy-eicosa-5(Z),14(Z)-dienoic acid, to enhance survival and protect against apoptosis in BPAECs stressed with serum starvation. 20-HETE produced a concentration-dependent increase in numbers of starved BPAECs and increased 5-bromo-2'-deoxyuridine incorporation. Caspase-3 activity, nuclear fragmentation studies, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays supported protection from apoptosis and enhanced survival of starved BPAECs treated with a single application of 20-HETE. Protection from apoptosis depended on intact NADPH oxidase, phosphatidylinositol 3 (PI3)-kinase, and ROS production. 20-HETE-stimulated ROS generation by BPAECs was blocked by inhibition of PI3-kinase or Akt activity. These data suggest 20-HETE-associated protection from apoptosis in BPAECs required activation of PI3-kinase and Akt and generation of ROS. 20-HETE also protected against apoptosis in BPAECs stressed by lipopolysaccharide, and in mouse PAs exposed to hypoxia reoxygenation ex vivo. In summary, 20-HETE may afford a survival advantage to BPAECs through activation of prosurvival PI3-kinase and Akt pathways, NADPH oxidase activation, and NADPH oxidase-derived superoxide.

Role of JNK in network formation of human lung microvascular endothelial cells.

The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or l-stereoisomer (l-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.

Emerging mechanisms for growth and protection of the vasculature by cytochrome P450-derived products of arachidonic acid and other eicosanoids.

Arachidonic acid (AA) is an essential fatty acid that is metabolized by cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 (CYP) enzymes to generate eicosanoids which in turn mediate a number of biological activities including regulation of angiogenesis. While much information on the effects of COX and LOX products is known, the physiological relevance of the CYP-derived products of AA are less well understood. CYP enzymes are highly expressed in the liver and kidney, but have also been detected at lower levels in the brain, heart and vasculature. A number of these enzymes, including members of the CYP 4 family, predominantly catalyze conversion of AA to 20-hydroxyeicosatetraenoic acid (20-HETE) while the CYP epoxygenases generate mainly epoxyeicosatrienoic acids (EETs). This review will focus on the emerging roles of inhibitors of eicosanoid production with emphasis on the CYP pathways, in the regulation of angiogenesis and tumor growth. We also discuss current observations describing the protective effects of EETs for survival of the endothelium.

Protective effects of epoxyeicosatrienoic acids on human endothelial cells from the pulmonary and coronary vasculature.

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 (CYP) metabolites synthesized from the essential fatty acid arachidonic acid to generate four regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET. Cultured human coronary artery endothelial cells (HCAECs) contain endogenous EETs that are increased by stimulation with physiological agonists such as bradykinin. Because EETs are known to modulate a number of vascular functions, including angiogenesis, we tested each of the four regioisomers to characterize their effects on survival and apoptosis of HCAECs and cultured human lung microvascular endothelial cells (HLMVECs). A single application of physiologically relevant concentration of 14,15-, 11,12-, and 8,9-EET but not 5,6-EET (0.75-300 nM) promoted concentration-dependent increase in cell survival of HLMVECs and HCAECs after removal of serum. The lipids also protected the same cells from death via the intrinsic, as well as extrinsic, pathways of apoptosis. EETs did not increase intracellular calcium concentration ([Ca2+]i) or phosphorylate mitogen-activated protein kinase p44/42 when applied to these cells, and their protective action was attenuated by the phosphotidylinositol-3 kinase inhibitor wortmannin (10 microM) but not the cyclooxygenase inhibitor indomethacin (20 microM). Our results demonstrate for the first time the capacity of EETs to enhance human endothelial cell survival by inhibiting both the intrinsic, as well as extrinsic, pathways of apoptosis, an important underlying mechanism that may promote angiogenesis and endothelial survival during atherosclerosis and related cardiovascular ailments.