Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth.

Colorectal cancer is the third leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Around 20% colon cancer cases are closely linked with colitis. Both environmental and genetic factors are thought to contribute to colon inflammation and tumor development. However, the genetic factors regulating colitis and colon tumorigenesis remain elusive. Since reactive oxygen species (ROS) is vitally involved in tissue inflammation and tumorigenesis, here we employed a genome-wide CRISPR knockout screening approach to systemically identify the genetic factors involved in the regulation of oxidative stress. Next generation sequencing (NGS) showed that over 600 gRNAs including the ones targeting LGALS2 were highly enriched in cells survived after sublethal H2O2 challenge. LGALS2 encodes the glycan-binding protein Galectin 2 (Gal2), which is predominantly expressed in the gastrointestinal tract and downregulated in human colon tumors. To examine the role of Gal2 in colitis, we employed the dextran sodium sulfate (DSS)-induced acute colitis model in mice with (WT) or without Lgals2 (Gal2-KO) and showed that Gal2 deficiency ameliorated DSS-induced colitis. We further demonstrated that Gal2-KO mice developed significantly larger tumors than WT mice using Azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal cancer model. We found that STAT3 phosphorylation was significantly increased in Gal2-deficient tumors as compared to those in WT mice. Gal2 overexpression decreased the proliferation of human colon tumor epithelial cells and blunted H2O2-induced STAT3 phosphorylation. Overall, our results demonstrate that Gal2 plays a suppressive role in colon tumor growth and highlights the therapeutic potential of Gal2 in colon cancer.

Genetic disruption of the inflammasome adaptor ASC has minimal impact on the pathogenesis of Duchenne muscular dystrophy in mdx mice.

AIM: Up-regulation of inflammasome proteins was reported in dystrophin-deficient muscles. However, it remains to be determined whether inflammasome activation plays a role in the pathogenesis of Duchenne muscular dystrophy. This study was therefore set out to investigate whether genetic disruption of the inflammasome pathway impacts the disease progression in mdx mice. MAIN METHODS: Mice deficient in both dystrophin and ASC (encoded by Pycard [PYD And CARD Domain Containing]) were generated. The impact of ASC deficiency on muscular dystrophy of mdx mice were assessed by measurements of serum cytokines, Western blot, real-time PCR and histopathological staining. KEY FINDINGS: The pro-inflammatory cytokines such as TNF-α, IL-6, KC/GRO and IL-10 were markedly increased in the sera of 8-week-old mdx mice compared to WT. Western blotting showed that P2X7, caspase-1, ASC and IL-18 were upregulated. Disruption of ASC and dystrophin expression in the mdx/ASC-/- mice was verified by Western blot analysis. Histopathological analysis did not find significant alterations in the muscular dystrophy phenotype in mdx/ASC-/- mice as compared to mdx mice. SIGNIFICANCE: Taken together, our results show that disruption of the central adaptor ASC of the inflammasome is insufficient to alleviate muscular dystrophy phenotype in mdx mice.

Correction: Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Life-Long AAV-Mediated CRISPR Genome Editing in Dystrophic Heart Improves Cardiomyopathy without Causing Serious Lesions in mdx Mice.

Previous studies from others and us have demonstrated that CRISPR genome editing could offer a promising therapeutic strategy to restore dystrophin expression and function in the skeletal muscle and heart of Duchenne muscular dystrophy (DMD) mouse models. However, the long-term efficacy and safety of CRISPR genome-editing therapy for DMD has not been well established. We packaged both SaCas9 and guide RNA (gRNA) together into one AAVrh.74 vector, injected two such vectors (targeting intron 20 and intron 23, respectively) into mdx pups at day 3 and evaluated the mice at 19 months. We found that AAVrh.74-mediated life-long CRISPR genome editing in mdx mice restored dystrophin expression and improved cardiac function without inducing serious adverse effects. PCR analysis and targeted deep sequencing showed that the DSBs were mainly repaired by the precise ligation of the two cut sites. Serological and histological examination of major vital organs did not reveal any signs of tumor development or other deleterious defects arising from CRISPR genome editing. These results support that in vivo CRISPR genome editing could be developed as a safe therapeutic treatment for DMD and potentially other diseases.

Automated muscle histopathology analysis using CellProfiler.

BACKGROUND: Histological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and centrally nucleated muscle fibers. These parameters offer insights into the dynamic adaptation of skeletal muscle cells during repeated cycles of degeneration and regeneration associated with many muscle diseases and injuries. Computational programs designed to obtain these parameters would greatly facilitate such efforts and offer significant advantage over manual image analysis, which is very labor-intensive and often subjective. Here, we describe a customized pipeline termed MuscleAnalyzer for muscle histology analysis based upon CellProfiler, a free, open-source software for measuring and analyzing cell images. RESULTS: The MuscleAnalyzer pipeline consists of loading, adjusting, and running a series of image-processing modules provided by CellProfiler. This pipeline was evaluated using wild-type and mdx muscle sections co-stained with laminin (to demarcate the muscle fiber boundaries) and 4',6-diamidino-2-phenylindole (DAPI, to label the nuclei). The immunofluorescence images analyzed using the MuscleAnalyzer pipeline or manually yielded similar results in the number of muscle fibers per image (p = 0.42) and central nucleated fiber (CNF) percentage (p = 0.29) in mdx mice. However, for a total of 67 images, CellProfiler completed the analysis in ~ 10 min on a regular PC while it took an investigator ~ 3 h using the manual approach in order to quantify the number of muscle fibers and CNF. Moreover, the MuscleAnalyzer pipeline also provided the measurement of the cross-sectional area (CSA) and minimal Feret's diameter (MFD) of muscle fibers, and thus fiber size distribution can be plotted. CONCLUSIONS: Our data indicate that the MuscleAnalyzer pipeline can efficiently and accurately analyze laminin and DAPI co-stained muscle images in a batch format and provide quantitative measurements for muscle histological properties such as muscle fiber diameters, fiber size distribution, and CNF percentage.

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies.This article has an associated First Person interview with the first author of the paper.

Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations.

Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5 (ANO5), which belongs to the anoctamin protein family. Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes; instead, one of these mice was observed to present with some abnormality in sperm motility. In contrast, a third line of ANO5-knockout (KO) mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology. Many of the ANO5-related patients carry point mutations or small insertions/deletions (indels) in the ANO5 gene. To more closely mimic the human ANO5 mutations, we engineered mutant ANO5 rabbits via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.

3',4'-dihydroxyflavonol ameliorates endoplasmic reticulum stress-induced apoptosis and endothelial dysfunction in mice.

Endoplasmic reticulum (ER) stress has been implicated in the development of hypertension 3 through the induction of endothelial impairment. As 3',4'-dihydroxyflavonol (DiOHF) 4 reduces vascular injury caused by ischaemia/reperfusion or diabetes, and flavonols have been demonstrated to attenuate ER stress, we investigated whether DiOHF can protect mice from ER stress-induced endothelial dysfunction. Male C57BLK/6 J mice were injected with tunicamycin to induce ER stress in the presence or absence of either DiOHF or tauroursodeoxycholic acid (TUDCA), an inhibitor of ER stress. Tunicamycin elevated blood pressure and impaired endothelium-dependent relaxation. Moreover, in aortae there was evidence of ER stress, oxidative stress and reduced NO production. This was coincident with increased NOX2 expression and reduced phosphorylation of endothelial nitric oxide synthase (eNOS) on Ser1176. Importantly, the effects of tunicamycin were significantly ameliorated by DiOHF or TUDCA. DiOHF also inhibited tunicamycin-induced ER stress and apoptosis in cultured human endothelial cells (HUVEC). These results provide evidence that ER stress is likely an important initiator of endothelial dysfunction through the induction of oxidative stress and a reduction in NO synthesis and that DiOHF directly protects against ER stress- induced injury. DiOHF may be useful to prevent ER and oxidative stress to preserve endothelial function, for example in hypertension.

Paeonol Attenuates LPS-Induced Endothelial Dysfunction and Apoptosis by Inhibiting BMP4 and TLR4 Signaling Simultaneously but Independently.

Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.

Angiotensin II Causes ß-Cell Dysfunction Through an ER Stress-Induced Proinflammatory Response.

The metabolic syndrome is associated with an increase in the activation of the renin angiotensin system, whose inhibition reduces the incidence of new-onset diabetes. Importantly, angiotensin II (AngII), independently of its vasoconstrictor action, causes ß-cell inflammation and dysfunction, which may be an early step in the development of type 2 diabetes. The aim of this study was to determine how AngII causes ß-cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurine-conjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and ß-cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal ß cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes ß-cell dysfunction in murine islets of Langerhans both in vivo and ex vivo. These effects were significantly attenuated by TUDCA, an inhibitor of ER stress. We also show that AngII-induced ER stress is required for the increased expression of proinflammatory cytokines and is caused by reactive oxygen species and IP3 receptor activation. These data reveal that the induction of ER stress is critical for AngII-induced ß-cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes.

Chronic treatment with paeonol improves endothelial function in mice through inhibition of endoplasmic reticulum stress-mediated oxidative stress.

Endoplasmic reticulum (ER) stress leads to endothelial dysfunction which is commonly associated in the pathogenesis of several cardiovascular diseases. We explored the vascular protective effects of chronic treatment with paeonol (2'-hydroxy-4'-methoxyacetophenone), the major compound from the root bark of Paeonia suffruticosa on ER stress-induced endothelial dysfunction in mice. Male C57BL/6J mice were injected intraperitoneally with ER stress inducer, tunicamycin (1 mg/kg/week) for 2 weeks to induce ER stress. The animals were co-administered with or without paeonol (20 mg/kg/oral gavage), reactive oxygen species (ROS) scavenger, tempol (20 mg/kg/day) or ER stress inhibitor, tauroursodeoxycholic acid (TUDCA, 150 mg/kg/day) respectively. Blood pressure and body weight were monitored weekly and at the end of treatment, the aorta was isolated for isometric force measurement. Protein associated with ER stress (GRP78, ATF6 and p-eIF2α) and oxidative stress (NOX2 and nitrotyrosine) were evaluated using Western blotting. Nitric oxide (NO) bioavailability were determined using total nitrate/nitrite assay and western blotting (phosphorylation of eNOS protein). ROS production was assessed by en face dihydroethidium staining and lucigenin-enhanced chemiluminescence assay, respectively. Our results revealed that mice treated with tunicamycin showed an increased blood pressure, reduction in body weight and impairment of endothelium-dependent relaxations (EDRs) of aorta, which were ameliorated by co-treatment with either paeonol, TUDCA and tempol. Furthermore, paeonol reduced the ROS level in the mouse aorta and improved NO bioavailability in tunicamycin treated mice. These beneficial effects of paeonol observed were comparable to those produced by TUDCA and tempol, suggesting that the actions of paeonol may involve inhibition of ER stress-mediated oxidative stress pathway. Taken together, the present results suggest that chronic treatment with paeonol preserved endothelial function and normalized blood pressure in mice induced by tunicamycin in vivo through the inhibition of ER stress-associated ROS.

Renal targeting potential of a polymeric drug carrier, poly-l-glutamic acid, in normal and diabetic rats.

BACKGROUND AND PURPOSE: Poly-l-glutamic acid (PG) has been used widely as a carrier to deliver anticancer chemotherapeutics. This study evaluates PG as a selective renal drug carrier. EXPERIMENTAL APPROACH: 3H-deoxycytidine-labeled PGs (17 or 41 kDa) and 3H-deoxycytidine were administered intravenously to normal rats and streptozotocin-induced diabetic rats. The biodistribution of these compounds was determined over 24 h. Accumulation of PG in normal kidneys was also tracked using 5-(aminoacetamido) fluorescein (fluoresceinyl glycine amide)-labeled PG (PG-AF). To evaluate the potential of PGs in ferrying renal protective anti-oxidative stress compounds, the model drug 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) was conjugated to 41 kDa PG to form PG-AEBSF. PG-AEBSF was then characterized and evaluated for intracellular anti-oxidative stress efficacy (relative to free AEBSF). RESULTS: In the normal rat kidneys, 17 kDa radiolabeled PG (PG-Tr) presents a 7-fold higher, while 41 kDa PG-Tr shows a 15-fold higher renal accumulation than the free radiolabel after 24 h post injection. The accumulation of PG-AF was primarily found in the renal tubular tissues at 2 and 6 h after an intravenous administration. In the diabetic (oxidative stress-induced) kidneys, 41 kDa PG-Tr showed the greatest renal accumulation of 8-fold higher than the free compound 24 h post dose. Meanwhile, the synthesized PG-AEBSF was found to inhibit intracellular nicotinamide adenine dinucleotide phosphate oxidase (a reactive oxygen species generator) at an efficiency that is comparable to that of free AEBSF. This indicates the preservation of the anti-oxidative stress properties of AEBSF in the conjugated state. CONCLUSION/IMPLICATIONS: The favorable accumulation property of 41 kDa PG in normal and oxidative stress-induced kidneys, along with its capabilities in conserving the pharmacological properties of the conjugated renal protective drugs, supports its role as a potential renal targeting drug carrier.

Sodium nitrite exerts an antihypertensive effect and improves endothelial function through activation of eNOS in the SHR.

Sodium nitrite (NaNO2) induces relaxation in isolated arteries partly through an endothelium-dependent mechanism involving NO-eNOS-sGC-cGMP pathway. The present study was designed to investigate the effect of chronic NaNO2 administration on arterial systolic blood pressure (SBP) and vascular function in hypertensive rats. NaNO2 (150 mg L-1) was given in drinking water for four weeks to spontaneously (SHR) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) treated hypertensive SD rats. Arterial SBP and vascular function in isolated aortae were studied. Total plasma nitrate/nitrite and vascular cyclic guanosine monophosphate (cGMP) levels were measured using commercially available assay kits. Vascular nitric oxide (NO) levels were evaluated by DAF-FM fluorescence while the proteins involved in endothelial nitric oxide synthase (eNOS) activation was determined by Western blotting. NaNO2 treatment reduced SBP, improved the impaired endothelium-dependent relaxation, increased plasma total nitrate/nitrite level and vascular tissue NO and cGMP levels in SHR. Furthermore, increased presence of phosphorylated eNOS and Hsp-90 was observed in NaNO2-treated SHR. The beneficial effect of nitrite treatment was not observed in L-NAME treated hypertensive SD rats. The present study provides evidence that chronic treatment of genetically hypertensive rats with NaNO2 improves endothelium-dependent relaxation in addition to its antihypertensive effect, partly through mechanisms involving activation of eNOS.

Paeonol protects against endoplasmic reticulum stress-induced endothelial dysfunction via AMPK/PPARδ signaling pathway.

Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1µM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5µg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.

Angiotensin 1-7 Protects against Angiotensin II-Induced Endoplasmic Reticulum Stress and Endothelial Dysfunction via Mas Receptor.

Angiotensin 1-7 (Ang 1-7) counter-regulates the cardiovascular actions of angiotensin II (Ang II). The present study investigated the protective effect of Ang 1-7 against Ang II-induced endoplasmic reticulum (ER) stress and endothelial dysfunction. Ex vivo treatment with Ang II (0.5 µM, 24 hours) impaired endothelium-dependent relaxation in mouse aortas; this harmful effect of Ang II was reversed by co-treatment with ER stress inhibitors, l4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) as well as Ang 1-7. The Mas receptor antagonist, A779, antagonized the effect of Ang 1-7. The elevated mRNA expression of CHOP, Grp78 and ATF4 or protein expression of p-eIF2α and ATF6 (ER stress markers) in Ang II-treated human umbilical vein endothelial cells (HUVECs) and mouse aortas were blunted by co-treatment with Ang 1-7 and the latter effect was reversed by A779. Furthermore, Ang II-induced reduction in both eNOS phosphorylation and NO production was inhibited by Ang 1-7. In addition, Ang 1-7 decreased the levels of ER stress markers and augmented NO production in HUVECs treated with ER stress inducer, tunicamycin. The present study provides new evidence for functional antagonism between the two arms of the renin-angiotensin system in endothelial cells by demonstrating that Ang 1-7 ameliorates Ang II-stimulated ER stress to raise NO bioavailability, and subsequently preserves endothelial function.

Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway.

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma "antihypertensive tea" is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), N(G)-nitro-L-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity.

Sodium nitrite causes relaxation of the isolated rat aorta: By stimulating both endothelial NO synthase and activating soluble guanylyl cyclase in vascular smooth muscle.

Ingestion of dietary nitrites lowers arterial blood pressure in experimental animals and in humans. However, the exact mechanism underlying the hypotensive effect of nitrite remains unclear. The present study compared nitrite-induced responses in rings (with or without endothelium) of aortae of 18-20weeks old Wistar-Kyoto Rats (WKY) and spontaneously hypertensive (SHR) rats and investigated the underlying mechanism. Relaxations of aortae from WKY and SHR to increasing concentrations (1nM-100µM) of sodium nitrite (NaNO2) were determined during sustained contractions to phenylephrine, in the absence and presence of pharmacological agents. The nitrite-induced relaxations were concentration-dependent and larger in SHR than in WKY aortic rings. Inhibition of endothelial nitric oxide synthase (eNOS) and the absence of endothelium decreased nitrite-induced relaxations in both WKY and SHR aortae, indicating the role of endothelium-derived nitric oxide (NO) in the response. The involvement of eNOS was further confirmed by increases in phosphorylation of eNOS at ser1177 in HUVEC cells following treatment with sodium nitrite. The presence of NO scavengers decreased the relaxation to nitrite in both WKY and SHR preparations while inhibition of soluble guanylyl cyclase (sGC) abolished the response, indicating that besides producing NO, nitrite also induces relaxation by directly activating the enzyme. Thus, the present study demonstrates that the sensitivity to exogenous nitrite is increased in the aorta of the SHR compared to that of the WKY. The endothelium-dependent component of the relaxation to nitrite involves activation of eNOS with production of endothelium-derived NO, while the endothelium-independent component is due to stimulation of sGC.

Des-aspartate angiotensin I (DAA-I) reduces endothelial dysfunction in the aorta of the spontaneously hypertensive rat through inhibition of angiotensin II-induced oxidative stress.

Des-aspartate angiotensin I (DAA-I), an endogenous nonapeptide, counteracts several effects of angiotensin II on vascular tone. The aim of this study was to investigate the acute protective effect of DAA-I on endothelial function in the spontaneously hypertensive rat (SHR) as well as its effect on angiotensin II-induced contractions and oxidative stress. Aortic rings were incubated with DAA-I (0.1µM) for 30min prior to the assessment of angiotensin II-induced contractions (0.1nM-10µM) in WKY and SHR aortas. Total nitrate and nitrite levels were assessed using a colorimetric method and reactive oxygen species (ROS) were measured by dihydroethidium (DHE) fluorescence and lucigenin-enhanced chemiluminescence. The effect of DAA-I was also assessed against endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, respectively. Angiotensin II-induced contractions were significantly reduced by DAA-I, losartan and tempol. Incubation with ODQ (soluble guanylyl cyclase inhibitor) and removal of the endothelium prevented the reduction of angiotensin II-induced contractions by DAA-I. Total nitrate and nitrite levels were increased in DAA-I, losartan and tempol treated-SHR tissues while ROS level was reduced by DAA-I and the latter inhibitors. In addition, DAA-I significantly improved the impaired acetylcholine-induced relaxation in SHR aortas whilst sodium nitroprusside-induced endothelium-independent relaxation remained unaffected. The present findings indicate that improvement of endothelial function by DAA-I in the SHR aorta is mediated through endothelium-dependent release of nitric oxide and inhibition of angiotensin II-induced oxidative stress.