Evaluation of self-amplifying mRNA platform for protein expression and genetic stability: Implication for mRNA therapies.

The consecutive launch of mRNA vaccines like mRNA-1273, BNT 162b2, and GEMCOVAC®-19 against COVID-19 has triggered the debate of long-term expression, safety, and genomic integration of the mRNA vaccine platforms. In the present study, we examined the longevity of antigenic protein expression of mRNA-614 and mRNA-S1LC based on self-amplifying mRNA (SAM) in Expi-293F™, HEK-293 T, and ARPE-19 cells. The protein expression was checked by sandwich-ELISA, FACS, luciferase activity assay, and Western blot. The transcribed antigenic mRNA was sequenced and found to be un-mutated. Additionally, no genomic integration of the reverse transcribed mRNA was observed even up to 7 days post-transfection as verified by PCR. Furthermore, we have generated high-quality 3D structures of non-structural proteins (nsPs) in silico and the genes encoding for the nsPs were cloned and expressed using the T7 system. Findings from the current study have strengthened the fact that the alphavirus-based SAM platform has the potential to become a modality in the upcoming years.

Glycemic, insulinemic and methylglyoxal postprandial responses to starches alone or in whole diets in dogs versus cats: Relating the concept of glycemic index to metabolic responses and gene expression.

Species differences between domestic cats (Felis catus) and dogs (Canis familiaris) has led to differences in their ability to digest, absorb and metabolize carbohydrates through poorly characterized mechanisms. The current study aimed to first examine biopsied small intestine, pancreas, liver and skeletal muscle from laboratory beagles and domestic cats for mRNA expression of key enzymes involved in starch digestion (amylase), glucose transport (sodium-dependent SGLTs and -independent glucose transporters, GLUT) and glucose metabolism (hexokinase and glucokinase). Cats had lower mRNA expression of most genes examined in almost all tissues compared to dogs (p < 0.05). Next, postprandial glucose, insulin, methylglyoxal (a toxic glucose metabolite) and d-lactate (metabolite of methylglyoxal) after single feedings of different starch sources were tested in fasted dogs and cats. After feeding pure glucose, peak postprandial blood glucose and methylglyoxal were surprisingly similar between dogs and cats, except cats had a longer time to peak and a greater area under the curve consistent with lower glycolytic enzyme expression. After feeding starches or whole diets to dogs, postprandial glycemic response, glycemic index, insulin, methylglyoxal and d-lactate followed reported glycemic index trends in humans. In contrast, cats showed very low to negligible postprandial glycemic responses and low insulin after feeding different starch sources, but not whole diets, with no relationship to methylglyoxal or d-lactate. Thus, the concept of glycemic index appears valid in dogs, but not cats. Differences in amylase, glucose transporters, and glycolytic enzymes are consistent with species differences in starch and glucose handling between cats and dogs.

The effects of oral arginine on its metabolic pathways in Sprague-Dawley rats.

Oral arginine supplements are popular mainly for their presumed vasodilatory benefit. Arginine is a substrate for at least four enzymes including nitric oxide synthase (NOS) and arginase, but the impact of oral supplements on its different metabolic pathways is not clear. Deficiencies of arginine-metabolising enzymes are associated with conditions such as hyperammonaemia, endothelial dysfunction, central nervous system and muscle dysfunction, which complicate the use of oral arginine supplements. We examined the effect of l-arginine (l-Arg) and d-arginine (d-Arg), each at 500 mg/kg per d in drinking water administered for 4 weeks to separate groups of 9-week-old male Sprague-Dawley rats. We quantified the expression of enzymes and plasma, urine and organ levels of various metabolites of arginine. l-Arg significantly decreased cationic transporter-1 expression in the liver and the ileum and increased endothelial NOS expression in the aorta and the kidney and plasma nitrite levels, but did not affect the mean arterial pressure. l-Arg also decreased the expression of arginase II in the ileum, arginine:glycine amidinotransferase in the liver and the kidney and glyoxalase I in the liver, ileum and brain, but increased the expression of arginine decarboxylase and polyamines levels in the liver. d-Arg, the supposedly inert isomer, also unexpectedly affected the expression of some enzymes and metabolites. In conclusion, both l- and d-Arg significantly affected enzymes and metabolites in several pathways that use arginine as a substrate and further studies with different doses and treatment durations are planned to establish their safety or adverse effects to guide their use as oral supplements.

The Small Molecule Indirubin-3'-Oxime Inhibits Protein Kinase R: Antiapoptotic and Antioxidant Effect in Rat Cardiac Myocytes.

Double-stranded, RNA-dependent protein kinase R (PKR) is a serine/threonine protein kinase activated by various stress signals. It plays an important role in inflammation, insulin sensitivity and glucose homeostasis. Increased PKR activity has been observed in obese humans as well as in obese diabetic mice. Indirubin-3'-oxime (I3O) is an effective inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3-beta. However, the effects of I3O on PKR activity/expression in cultured rat cardiomyocytes have not been reported. We investigated whether I3O attenuates the effects of high glucose on PKR, oxidative stress and apoptotic gene markers. Quantitative PCR and western blotting were used to measure protein and mRNA, respectively. High glucose treatment caused significant increase in the PKR protein/mRNA expression, which was attenuated by co-treatment with I3O. High glucose-treated, cultured cardiomyocytes developed a significant increase in mRNA expression for c-Jun-N-terminal kinase, caspase-3 and NF-ĸB, which were all attenuated by pretreatment with I3O. There was also a significant increase in reactive oxygen species generation in high glucose-treated, cultured cardiomyocytes, which was attenuated by pretreatment with I3O. In conclusion, I3O may have a preventive role against the deleterious effects of high glucose in the heart.

The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.

Oral L-arginine supplements are popular mainly for their nitric oxide mediated vasodilation, but their physiological impact is not fully known. L-arginine is a substrate of several enzymes including arginase, nitric oxide synthase, arginine decarboxylase, and arginine: glycine amidinotransferase (AGAT). We have published a study on the physiological impact of oral L- and D-arginine at 500 mg/kg/day for 4 wks in male Sprague-Dawley rats. We investigated the effects of oral L-arginine and D-arginine at a higher dose of 1000 mg/kg/d for a longer treatment duration of 16 wks in 9-week-old male Sprague-Dawley rats. We measured the expression and activity of L-arginine metabolizing enzymes, and levels of their metabolites in the plasma and various organs. L-arginine did not affect the levels of L-arginine and L-lysine in the plasma and various organs. L-arginine decreased arginase protein expression in the upper small intestine, and arginase activity in the plasma. It also decreased AGAT protein expression in the liver, and creatinine levels in the urine. L-arginine altered arginine decarboxylase protein expression in the upper small intestine and liver, with increased total polyamines plasma levels. Endothelial nitric oxide synthase protein was increased with D-arginine, the presumed metabolically inert isomer, but not L-arginine. In conclusion, oral L-arginine and D-arginine at a higher dose and longer treatment duration significantly altered various enzymes and metabolites in the arginine metabolic pathways, which differed from alterations produced by a lower dose shorter duration treatment published earlier. Further studies with differing doses and duration would allow for a better understanding of oral L-arginine uses, and evidence based safe and effective dose range and duration.

Arginine attenuates methylglyoxal- and high glucose-induced endothelial dysfunction and oxidative stress by an endothelial nitric-oxide synthase-independent mechanism.

Methylglyoxal (MG), a reactive metabolite of glucose, has high affinity for arginine and is a precursor of advanced glycation endproducts (AGEs). We tested the hypothesis that L-arginine, and its inactive isomer D-arginine, can efficiently scavenge MG, administered exogenously or produced endogenously from high glucose, and attenuate its harmful effects including endothelial dysfunction and oxidative stress by an endothelial nitric-oxide synthase (eNOS)-independent mechanism. We used isolated aortic rings from 12-week-old male Sprague-Dawley rats and cultured human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs). Both D-arginine and L-arginine prevented the attenuation of acetylcholine-induced endothelium-dependent vasorelaxation by MG and high glucose. However, the inhibitory effect of the NOS inhibitor N(ω)-nitro-L-arginine methyl ester on vasorelaxation was prevented by L-arginine, but not D-arginine. MG and high glucose increased protein expression of arginase, a novel finding, NADPH oxidase 4, and nuclear factor κB and increased production of reactive oxygen species in HUVECs and VSMCs, which were attenuated by D-arginine and L-arginine. However, D-arginine and L-arginine did not attenuate MG- and high glucose-induced increased arginase activity in VSMCs and the aorta. D-arginine and L-arginine also attenuated the increased formation of the MG-specific AGE N(ε)-carboxyethyl lysine, caused by MG and high glucose in VSMCs. In conclusion, arginine attenuates the increased arginase expression, oxidative stress, endothelial dysfunction, and AGE formation induced by MG and high glucose by an eNOS-independent mechanism. The therapeutic potential of arginine against MG- and high glucose-induced pathology merits further investigation.

Human gingival mesenchymal stem cells retain their growth and immunomodulatory characteristics independent of donor age.

Aging has been reported to deteriorate the quantity and quality of mesenchymal stem cells (MSCs), which affect their therapeutic use in regenerative medicine. A dearth of age-related stem cell research further restricts their clinical applications. The present study explores the possibility of using MSCs derived from human gingival tissues (GMSCs) for studying their ex vivo growth characteristics and differentiation potential with respect to donor age. GMSCs displayed decreased in vitro adipogenesis and in vitro and in vivo osteogenesis with age, but in vitro neurogenesis remained unaffected. An increased expression of p53 and SIRT1 with donor age was correlated to their ability of eliminating tumorigenic events through apoptosis or autophagy, respectively. Irrespective of donor age, GMSCs displayed effective immunoregulation and regenerative potential in a mouse model of LPS-induced acute lung injury. Thus, we suggest the potential of GMSCs for designing cell-based immunomodulatory therapeutic approaches and their further extrapolation for acute inflammatory conditions such as acute respiratory distress syndrome and COVID-19.

Vasoactive Effects of Acute Ergot Exposure in Sheep.

Ergotism is a common and increasing problem in Saskatchewan's livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α1-adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α1-adrenergic agonist) was compared between the two groups before and after TE (α1-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC50 = 1.74 × 10-6 M; Exp EC50 = 1.079 × 10-6 M, p = 0.046). TE treatment resulted in a significant dose-dependent increase in EC50 in both exposure and control groups (p < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, p = 0.36; TE 100 nM, p < 0.001; TE 300 nM, p < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α1-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.

Decreases in splanchnic vascular resistance contribute to hypotensive effects of L-serine in hypertensive rats.

l-Serine administration reduces mean arterial pressure (MAP) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats rendered hypertensive by chronic oral treatment with the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). To determine if the fall in MAP was due to decreases in vascular resistance or cardiac output (CO), and to record regional hemodynamic effects, we measured the distribution of fluorescent microspheres to single bolus intravenous injections of l-serine (1 mmol/kg) in 14-wk-old male WKY, SHR, and l-NAME-treated WKY rats. MAP and total peripheral resistance (TPR) were significantly higher (P < 0.01), whereas CO was lower in l-NAME-treated WKY (P < 0.01) and SHR (P < 0.05). l-Serine administration led to a rapid fall in MAP (WKY 22%, l-NAME-WKY 46%, SHR 34%,) and TPR (WKY 24%, l-NAME-WKY 68%, SHR 53%), whereas CO was elevated. In WKY rats, l-serine induced an increase in blood flow only in the small intestine (53%) while it was more profound in several vascular beds of hypertensive rats [l-NAME-WKY: small intestine (238%), spleen (184%), diaphragm (85%), and liver (65%); SHR: small intestine (217%), spleen (202%), diaphragm (116%), large intestine (105%), pancreas (96%), and liver (93%)]. Pretreatment with a combination of apamin (a small calcium-activated potassium channel inhibitor) and charybdotoxin (an intermediate calcium-activated potassium channel inhibitor) abolished the l-serine-induced changes in blood flow and TPR. l-Serine acts predominantly on apamin- and charybdotoxin-sensitive potassium channels in the splanchnic circulation to increase blood flow, thereby contributing to the fall in TPR and the pronounced blood pressure-lowering effect of l-serine in hypertensive rats.

Oxidative stress and aging: is methylglyoxal the hidden enemy?

Aging is a multifactorial process that involves changes at the cellular, tissue, organ and the whole body levels resulting in decreased functioning, development of diseases, and ultimately death. Oxidative stress is believed to be a very important factor in causing aging and age-related diseases. Oxidative stress is caused by an imbalance between oxidants such as reactive oxygen species (ROS) and antioxidants. ROS are produced from the mitochondrial electron transport chain and many oxidative reactions. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. MG levels are elevated in hyperglycemia and other conditions. An excess of MG formation can increase ROS production and cause oxidative stress. MG reacts with proteins, DNA and other biomolecules, and is a major precursor of advanced glycation end products (AGEs). AGEs are also associated with the aging process and age-related diseases such as cardiovascular complications of diabetes, neurodegenerative diseases and connective tissue disorders. AGEs also increase oxidative stress. In this review we discuss the potential role of MG in the aging process through increasing oxidative stress besides causing AGEs formation. Specific and effective scavengers and crosslink breakers of MG and AGEs are being developed and can become potential treatments to slow the aging process and prevent many diseases.

Attenuation of hypertension development by scavenging methylglyoxal in fructose-treated rats.

OBJECTIVES: Methylglyoxal is a reactive dicarbonyl intermediate of metabolism produced in the body. It reacts with certain proteins and forms damaging advanced glycation endproducts (AGEs) such as N epsilon-carboxyethyl-lysine (CEL) and N epsilon-carboxymethyl-lysine (CML). Increased methylglyoxal levels are found in diabetes mellitus and associated with hypertension development in the spontaneously hypertensive rats (SHR). The purpose of this study was to investigate whether increased endogenous formation of methylglyoxal and methylglyoxal-induced AGEs caused hypertension development in normotensive Sprague Dawley rats. METHODS: The rats were fed chronically for 16 weeks with fructose, a known precursor of methylglyoxal formation. One group of rats was cotreated with fructose and metformin, an AGEs formation inhibitor. Methylglyoxal and reduced glutathione (GSH) were measured by high performance liquid chromatography, whereas hydrogen peroxide was measured by a dicholorofluorescin assay. Immunohistochemistry was performed for endothelial nitric oxide synthase (eNOS), CEL and CML. RESULTS: Fructose-fed rats had elevated blood pressure, serum methylglyoxal and triglycerides and reduced serum levels of GSH. Methylglyoxal, hydrogen peroxide and CEL were increased in the aorta, whereas eNOS was reduced. CEL and CML were also increased in the mesenteric artery endothelium along with media/lumen ratio, signifying structural remodelling. All the harmful changes in fructose-fed rats were attenuated in metformin and fructose cotreated rats. CONCLUSION: Increased methylglyoxal, AGEs, oxidative stress and reduced eNOS along with structural remodeling of the vessel wall in the aorta and mesenteric artery likely play a role in the pathogenesis of hypertension.

L-Serine lowers while glycine increases blood pressure in chronic L-NAME-treated and spontaneously hypertensive rats.

OBJECTIVE: To determine the acute hemodynamic effects of the nonessential amino acid, glycine, and its precursor, L-serine, in normotensive and hypertensive rats. METHODS: Changes in mean arterial pressure and heart rate evoked by comparable intravenously administered doses (0.3-3.0 mmol/kg) of L-serine, D-serine and glycine were examined in anaesthetized normotensive 14-week-old male Sprague-Dawley, Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats and WKY rats subjected to chronic nitric oxide synthase inhibition by treatment with NG nitro L-arginine methyl ester (0.7 mg/ml in drinking water for 5 days). RESULTS: L-Serine evoked a greater maximal fall in mean arterial pressure [L-serine vs. D-serine in Sprague-Dawley rats, mean +/- standard error of the mean values (mmHg): 30 +/- 3 vs. 20 +/- 5, P < 0.05; in control WKY rats: 46 +/- 3 vs. 30 +/- 4, P < 0.05; in NG nitro L-arginine methyl ester-treated WKY rats: 93 +/- 6 vs. 41 +/- 5, P < 0.01; in spontaneously hypertensive rats: 81 +/- 7 vs. 39 +/- 5 P < 0.01]. The effects of L-serine were significantly reduced in rats pretreated with a combination of apamin and charybdotoxin, inhibitors of the small conductance and intermediate conductance calcium-activated potassium (KCa) channels. Glycine elicited a dose-dependent fall in mean arterial pressure in normotensive WKY rats (25 +/- 4; P < 0.01) and evoked pressor responses in both spontaneously hypertensive rats (29 +/- 3; P < 0.01) and NG nitro L-arginine methyl ester-pretreated hypertensive WKY (39 +/- 5; P < 0.01) rats. Both the depressor and pressor responses to glycine were abolished by pretreatment with the N-methyl D-aspartate receptor antagonist, MK-801. CONCLUSION: The profound stereo-selective antihypertensive effect of L-serine is neither mediated nor mimicked by glycine. It does not require N-methyl D-aspartate receptor activation by glycine but likely involves activation of endothelial KCa channels. L-Serine is a potential antihypertensive agent.

Chronic clofibrate administration prevents saline-induced endothelial dysfunction and oxidative stress in young Sprague-Dawley rats.

PURPOSE: High salt intake causes hypertension and endothelial dysfunction in young Sprague-Dawley rats. Clofibrate (clof) prevents this salt induced hypertension. We asked whether clof can prevent salt-induced endothelial dysfunction, and if so, its mechanism. We also questioned whether high salt intake can induce endothelial dysfunction without hypertension in older animals. METHODS: Young (Y, 5 weeks) and old (O, 53 weeks) male Sprague-Dawley rats were given either vehicle (Con, 20 mM Na2CO3) or 0.9% NaCl (Sal) to drink for three weeks. Some young rats received clof (80 mg/d) in their drinking fluid. After three weeks, we measured mean arterial pressure (MAP), endothelial function, by comparing hypotensive responses to acetylcholine (ACh, endothelium dependent) and sodium nitroprusside (SNP, endothelium independent), plasma total nitrite+nitrate levels (PNOx), by the Griess reaction, and aortic superoxide production by lucigenin chemiluminescence. RESULTS: Carotid artery MAP did not change in O. Sal-Y developed hypertension: 133+/-3 vs. 114+/-2 mmHg, P < 0.001, which was prevented by clof: 105+/-2 mmHg. ACh induced a similar dose dependent hypotensive response in Con-O and Sal-O that was inhibited by L-NAME (100mg/kg i.v.). Responses to ACh were blunted in Sal-Y but not in Con-Y. Further, L-NAME inhibited ACh responses only in Con-Y. The response to SNP was similar in all animals. Importantly, the ACh-induced hypotensive response was potentiated in clof+Sal-Y, an effect which was attenuated by blocking calcium-activated potassium channels (KCa) with a combination of apamin (50 ug/kg i.v.) + charybdotoxin (50 ug/kg i.v.), but not by L-NAME. PNOx was reduced in Sal-Y compared to Con-Y (2.09+/-0.26 vs. 4.8+/-0.35 microM, P < 0.001), but not in Sal-O. Aortic superoxide production was higher (P < 0.001) in Sal-Y (2388+/-40 milliunits/mg/min) than Sal-O (1107+/-159 milliunits/mg/min), but was reduced by clof (1378+/-64 milliunits/mg/min; P < 0.001). CONCLUSIONS: High salt intake increases oxidative stress in young animals, leading to impaired nitric oxide activity and endothelial dysfunction. Clofibrate prevents endothelial dysfunction partly through reduced O2 - formation but mainly via selective activation of KCa channels. Older animals are resistant to both salt induced hypertension and oxidative stress.

Free radical generation by methylglyoxal in tissues.

Methylglyoxal (MG) is a reactive dicarbonyl intermediate of the glycolytic pathway. Increased oxidative stress is associated with conditions of increased MG, such as diabetes mellitus. Increased oxidative stress is due to an increase in highly reactive by-products of metabolic pathways, the so-called reactive oxygen species, such as superoxide anion, hydroxyl radical, hydrogen peroxide, nitric oxide and peroxynitrite. These reactive species react with a variety of proteins, enzymes, lipids, DNA and other molecules and disrupt their normal function. Oxidative stress causes many pathological changes that lead to vascular complications of diabetes mellitus, hypertension, neurodegenerative diseases and aging. In this review we summarize the correlation of elevated MG and various reactive oxygen species, and the enzymes that produce them or take part in their disposal, such as antioxidant enzymes and cofactors. The findings reported in various studies reviewed have started filling in gaps in our knowledge that will ultimately provide us with a clear picture of how the whole process that causes cellular dysfunction is initiated.

Scaffold-Free Spheroids Derived from Stem Cells for Tissue-Engineering Applications.

Tissue engineering has gained attention in the past decade due to its efficient interaction with the host system and potential therapeutic capabilities. Although scaffold-based approaches provide much needed mechanical strength and support to the regenerating tissue, they also invite foreign body reaction initiated by macrophages, causing inflammation and toxicity, and may also sometime interfere with the regeneration of indigenous tissue due to very slow degradation. Therefore, spheroids provide a promising tool for improving cell survival and for preserving cell-to-cell interaction. They have promptly gained popularity because of their ability to provide superior cellular heterogeneity, nutrient and oxygen gradients (replicating the original tissue), matrix deposition, and gene expression profiles. Because of their ability to differentiate into multiple cell lineages, stem cell-based spheroids have opened new avenues for future regenerative medicine. In this review we focus on various methods for fabrication of spheroids from stem cells and their application in regenerative approaches for different tissues/organs.

Attenuation of hypertension development by aminoguanidine in spontaneously hypertensive rats: role of methylglyoxal.

BACKGROUND: Methylglyoxal (MG), a metabolite of glucose, and MG-induced advanced glycation endproducts (AGEs) are causatively associated with vascular complications of diabetes mellitus. We have previously reported elevated levels of MG and MG-induced AGEs in spontaneously hypertensive rats (SHR). The purpose of this study was to investigate the causative role of MG and MG-induced AGEs in the pathogenesis of hypertension in SHR. METHODS: Young SHR were treated with an AGE inhibitor, aminoguanidine, for 9 weeks. HPLC was used to determine plasma and aortic MG and reduced glutathione levels. The MG-induced AGEs, N epsilon-carboxyethyl-lysine (CEL) and argpyramidine, in the aorta were determined by immunohistochemistry. Vascular relaxation of small mesenteric arteries was measured using myograph. RESULTS: Chronic treatment with aminoguanidine attenuated age-dependent blood pressure (BP) increase in SHR. Plasma and aortic MG levels, and aortic levels of MG-induced AGEs, were significantly reduced after aminoguanidine treatment, which were comparable to those from age-matched Wistar Kyoto rats. Free radical level was significantly lowered, whereas reduced glutathione level was significantly increased by aminoguanidine treatment in the aortic tissues from SHR. Moreover, aminoguanidine therapy prevented the morphologic damage of vascular tissues in SHR and restored the endothelium-dependent relaxation to acetylcholine. Chronic aminoguanidine treatment also increased aortic endothelial nitric oxide synthase expression and reduced inducible nitric oxide synthase expression. CONCLUSIONS: The MG and MG-induced AGEs contribute to the pathogenesis of hypertension by altering the redox balance, causing vascular eutrophic inward remodeling, and inducing endothelial dysfunction in SHR.

Clofibrate acutely reverses saline-induced endothelial dysfunction: role of calcium-activated potassium channels.

BACKGROUND: Endothelium-dependent vascular relaxation is impaired in various disease states including hypertension. METHODS: We investigated whether a single bolus dose of clofibrate could rapidly reverse saline-induced endothelial dysfunction, in vivo, in salt-loaded Sprague-Dawley (S-D) rats. S-D rats, 5 weeks of age, were divided into two groups. One group served as a control (Con) and was given tap water; the other group (Sal) was given normal saline (0.9% NaCl) ad libitum for 3 weeks. RESULTS: Mean arterial pressure (MAP) was significantly higher (138 +/- 2 nu 112 +/- 2 mm Hg, P < .001), whereas the total plasma nitrite/nitrate levels were lower (1.7 +/- 0.3 v 2.8 +/- 0.2 micromol/L, P < .05) in Sal. At this time, endothelial function was assessed in vivo. Sal rats had decreased hypotensive responses to acetylcholine (ACh) but maintained normal responses to sodium nitroprusside. The ACh-induced hypotensive response was significantly inhibited by the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME, 100 mg kg(-1) intraperitoneally [ip]) only in Con rats. Clofibrate (Clof, 200 mg kg(-1) ip) did not change blood pressure but increased ACh-induced hypotensive responses only in Sal, an effect that was abolished by subsequent administration of apamin (Apa, 50 microg kg(-1) iv) and charybdotoxin (ChTx, 50 microg kg(-1) iv). Apa+ChTx blocked responses to ACh in Con and Sal, as expected. A single dose of clofibrate (200 mg kg(-1) ip), given subsequently to Apa+ChTx, restored responses to ACh in both the Con and Sal groups, again without affecting baseline MAP. CONCLUSION: Clofibrate has an acute salutary effect on endothelium-dependent vasodilation in saline-treated rats, probably mediated through vascular calcium-activated potassium channels and independent of an antihypertensive effect.

Inability to upregulate cytochrome P450 4A and 2C causes salt sensitivity in young Sprague-Dawley rats.

BACKGROUND: Young Sprague-Dawley rats develop high blood pressure (BP) when exposed to a high salt intake, whereas adult ones generally do not. We investigated the role of renal cytochromes P450 4A (CYP 4A) and 2C (CYP 2C) in maintaining normal BP. METHODS: Young (age 5 weeks) and adult (age 53 weeks) Sprague-Dawley rats were given either 20 mmol sodium carbonate (vehicle for clofibrate) or 0.9% saline to drink for 3 weeks. Some young animals received the peroxisome proliferator activated receptor (PPAR)alpha agonist clofibrate (80 mg daily). We measured tail-cuff and intra-arterial BP, weight change, sodium balance, 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (by high-performance liquid chromatography), and renal expression of CYP 4A and CYP 2C (by real-time reverse transcriptase-polymerase chain reaction). RESULTS: Saline-treated adult animals remained normotensive: systolic BP (SBP) 117 +/- 2 mm Hg v 117 +/- 1 mm Hg in control animals. In contrast, young rats given saline developed increased SBP: 134 +/- 2 mm Hg v 115 +/- 2 mm Hg in control animals (P < . 001). Interestingly, clofibrate lowered SBP to 102 +/- 2 mm Hg in saline-treated young rats but had no effect in control animals (114 +/- 2 mm Hg). Adult rats given saline increased renal expression of CYP 4A and 2C and excreted more 20-HETE. However, young rats given saline showed no induction, and even reduced CYP 4A and 2C, decreased urinary 20-HETE excretion, and retained sodium. Clofibrate increased renal CYP and 20-HETE excretion and prevented sodium retention. CONCLUSIONS: The products of renal CYP4A and 2C, including 20-HETE, aid in excreting salt. Animals that are unable to increase renal 20-HETE formation do not excrete sodium and are prone to hypertension.

EDHF-mediated rapid restoration of hypotensive response to acetylcholine after chronic, but not acute, nitric oxide synthase inhibition in rats.

Several in vitro studies have shown that endothelium-dependent vasodilatation is maintained by endothelium-derived hyperpolarizing factor (EDHF) or prostacyclin in vessels isolated from endothelial nitric oxide synthase knockout mice. Since this has not been addressed by in vivo studies, we sought to define the magnitude and the onset time of this compensation by recording blood pressure responses to endothelium-dependent vasodilators in rats treated acutely or chronically with the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME). Groups of male Sprague-Dawley rats were given plain water (control) or L-NAME (0.7 mg/ml) in drinking water for 1 day, 5 days, 3 wks or 6 wks. Dose-dependent hypotensive responses to acetylcholine, bradykinin and sodium nitroprusside were determined in anesthetized rats before and after acute intravenous infusion of either L-NAME or a combination of apamin plus charybdotoxin that would selectively inhibit EDHF. Acute L-NAME treatment increased the mean arterial pressure and inhibited acetylcholine- and bradykinin-induced fall in blood pressure in control but not in chronic L-NAME treated rats. The endothelium-dependent hypotensive responses to acetylcholine and bradykinin were restored in rats treated with L-NAME after a time period of 24 h along with increased sensitivity to sodium nitroprusside and reduced plasma nitrate+nitrite levels. While apamin+charybdotoxin pretreatment inhibited the responses to acetylcholine and bradykinin in both acute and chronic L-NAME treated groups, it was more pronounced in the latter group. In conclusion, chronic inhibition of nitric oxide synthase results in the development of a compensatory hypotensive response to acetylcholine within 24 h and this is mediated by EDHF.