Cyclosporine inhibits endotoxin-induced vasodilation of isolated rat resistance arterioles.

BACKGROUND: An isolated arteriole fails to dilate in response to endotoxin unless a segment of aorta is included in the perfusion system. The unknown substance released by the aorta after exposure to endotoxin is dependent upon the NF-kappaB pathway and induces inducible nitric oxide synthase (iNOS) in the arteriole. The purpose of this study was to determine if cyclosporine A (CSA) that inhibits both NF-kappaB and iNOS would prevent the vasodilatory response to endotoxin. MATERIALS AND METHODS: Rats were injected with either 10 mg/kg of CSA or oil vehicle followed by the removal of a cremaster muscle. The feeding arteriole was isolated from the cremaster and mounted on micropipettes and pressurized to 70 mmHg in a superfused tissue bath. After an hour equilibration to develop spontaneous tone, a 1 cm segment of aorta was placed in the superfusion system upstream from the arteriole and Salmonella enteriditis endotoxin was added to the buffer at a concentration of 2.5 microg/mL (ET) or continued infusion of buffer alone. Internal diameters of cannulated arterioles were measured with videomicroscopy and videocalipers for an additional hour. RESULTS: Arterioles downstream from an aorta exposed to vehicle but not endotoxin developed 22.8 +/- 3.7% tone that remained unchanged over the following hour. Arterioles exposed to endotoxin started with 22.5 +/- 2.8% spontaneous tone and this fell over the following hour to 11.8 +/- 3.6%, P < 0.05. Pre-treatment of the rats with CSA tended to increase resting tone and completely prevented the loss of tone after endotoxin. CONCLUSIONS: Pre-treatment of the aortic segment with CSA resulted in the development of increased tone in the downstream arteriole and completely blocked the vasodilatory response to endotoxin. These results suggest that CSA or a similar compound may be useful in the treatment of septic shock.

Endotoxin releases a substance from the aorta that dilates an isolated arteriole by up-regulating INOS.

BACKGROUND: Loss of vascular tone in resistance arterioles has been implicated as the cause of hypotension in septic shock. It is believed that the overproduction of nitric oxide (NO) by the inducible isoform of nitric oxide synthase (iNOS) results in the vasodilatation seen in septic shock. However, we have shown that endotoxin has no effect on vascular tone of an isolated resistance vessel unless the endotoxin flows over a segment of aorta or vena cava upstream in the superfusion line. The aim of this study was to determine if the subsequent vasodilation was due to the release of a direct vasodilator or production of NO in the arteriole and if its source was iNOS by using its selective inhibitor, aminoguanidine. MATERIALS AND METHODS: First-order rat cremaster arterioles (n = 36) were isolated and cannulated onto micropipettes, superfused with physiological buffer at 34 degrees C, pressurized to 70 mm Hg, and allowed to gain spontaneous tone over 90 min. A segment of abdominal aorta was then placed in series with the arteriole so that the superfusate passed over the aorta and then into the tissue bath containing the isolated arteriole. The vessels were allowed to equilibrate over 60 min. During this interval, the arteriole was exposed to l-NAME (100 mum), aminoguanidine (100 mum), or buffer. The aorta and arteriole were then superfused with endotoxin (Salmonella enteritidis 2.5 mug/ml). Internal diameters of cannulated arterioles were measured and recorded with videomicroscopy and videocalipers at a resolution of +/-1 mum every 15 min for 1 h. Six groups were created with n = 6 for each group: Group 1, endotoxin; Group 2, control; Group 3, l-NAME and endotoxin; Group 4, l-NAME; Group 5, aminoguanidine and endotoxin; and Group 6, aminoguanidine. RESULTS: After the 60-min equilibration period, there was no significant difference in resting tone among the six groups. At t = 120, the percentage of tone in the control group was 42.7 +/- 0.4% (mean +/- SEM) and this was not changed by treatment with aminoguanidine (42.2 +/- 0.7%). However, exposure to l-NAME alone resulted in vasoconstriction with a gain in tone to 49.5 +/- 1.6% (P > 0.05). Endotoxin alone caused arteriolar tone to fall to 33.5 +/- 1.2% (P < 0.05). Arterioles treated with aminoguanidine did not lose tone (42.6 +/- 1.7%) when exposed to endotoxin and arterioles treated with l-NAME retained their elevated tone (46.0 +/- 2.2%) after treatment with endotoxin. CONCLUSIONS: This study demonstrates that the aorta exposed to endotoxin releases a substance that vasodilates resistance arterioles through the up-regulation of iNOS. Aminoguanidine prevented the fall in tone following exposure to endotoxin, while use of the nonselective NOS inhibitor, l-NAME, not only blocked the fall due to endotoxin but increased basal tone by blocking the constitutively active eNOS.

Endotoxin releases a precursor to vasodilation from large veins in an NF-kappaB-dependent manner.

INTRODUCTION: Our previous studies have shown that when a segment of rat aorta was placed upstream and in series to a rat cremasteric isolated arteriole, endotoxin (ET) exposure produced significant vasodilatation. Without the aorta, no loss of tone was noted, indicating that a precursor, as of yet unidentified, was washed downstream, thereby inducing vasodilation. Prior treatment of the donor of either the aorta or the arteriole with pyrrolidine dithiocarbamate (PDTC), a potent NF-kappaB inhibitor, prevented the loss of tone. This suggests a role for NF-kappaB in the signaling pathway. The aim of this study was to determine if a large vein was also capable of releasing a similar factor in response to ET. MATERIALS AND METHODS: This project followed the same experimental design except that the upstream aortic segment was replaced by a segment of vena cava. Male Sprague-Dawley rats were given an intraperitoneal (ip) injection of PDTC (100 mg/kg) or a sham injection of saline. First-order cremasteric arterioles were isolated, cannulated, and pressurized. A segment of inferior vena cava was then placed in series with the microvascular preparation. Arterioles were allowed to equilibrate and achieve spontaneous myogenic tone in a bath of warm physiological buffer over 1 h (t = 0). Internal vessel diameters were measured with video calipers and the response to ET or continued infusion of buffer was measured over 2 h (t = 120). The control group (n = 8) received a sham injection and the vessels were exposed to buffer only. The ET group (n = 7) was exposed to ET only. The PDTC group (n = 5) received PDTC only. The PDTC/ET group (n = 6) received PDTC and was exposed to ET. RESULTS: After equilibration, spontaneous tone (measured as a percentage of maximal diameter) was similar in the four groups (t = 0). After 2 h (t = 120), the ET group had significantly less tone (30.1 +/- 3.6%; P < 0.05) than the control (44.8 +/- 2.6%), the PDTC group (43.0 +/- 1.3%), and the PDTC/ET group (49.4 +/- 3.0%). CONCLUSIONS: These results show that the vena cava is capable of releasing a factor leading to vasodilation in response to ET in a manner similar to the aorta. Produced by the veins, it will affect venous capacitance as well as contribute to the total amount in the plasma pool affecting the tone of the resistant arterioles. Thus, it appears that these large conducting vessels, regardless of origin, play a role in the deleterious effects during septic events.

Pioglitazone prevents hypertension and reduces oxidative stress in diet-induced obesity.

The objective of this study was to determine the effect of pioglitazone on blood pressure (BP) and oxidative balance in obese, hypertensive, Sprague-Dawley rats and to identify some of the molecular mechanisms involved. After 12 weeks of a moderately high-fat diet, rats diverged into obesity-prone (OP) and obesity-resistant (OR) groups (n=6 per group). At the end of the diet, peroxisome proliferator activated receptor-gamma (PPARgamma) mRNA expression and activity in the renal cortex and medulla of OP rats were significantly lower compared with that in OR rats. Pioglitazone treatment increased PPARgamma expression and activity in OP rats, suggesting a possible direct ligand-related effect of pioglitazone. As opposed to the untreated OP group, which showed moderate hypertension (systolic BP=159+/-5.3 mm Hg) after 12 weeks, pioglitazone-treated rats were normotensive (systolic BP=123.9+/-2.7 mm Hg). Insulin production was reduced by 2-fold in the OP group treated with pioglitazone. Urinary isoprostanes and renal lipid peroxides were also reduced in OP rats treated with pioglitazone compared with untreated counterparts. Also, expression of p47phox and gp91phox, both increased in OP versus OR rats, was reduced in the former by pioglitazone treatment. In addition, pioglitazone treatment increased nitrate/nitrite excretion and expression of renal endothelial and neuronal nitric oxide synthase. Collectively, the results show that pioglitazone treatment prevented hypertension and renal oxidative stress both by reducing free-radical production and by increasing nitric oxide production/availability.

Effect of salt on hypertension and oxidative stress in a rat model of diet-induced obesity.

High-salt diet is known to induce or aggravate hypertension in animal models of hypertension and in humans. When Sprague-Dawley rats (n = 60) are fed a moderately high-fat diet (32% kcal fat, 0.8% NaCl) for 10 wk, about one-half develop obesity [obesity prone (OP)] and mild hypertension, whereas the other half [obesity resistant (OR)] maintain body weight equivalent to a low-fat control (C) and are normotensive. The aim of this study was to test the effect of high-NaCl diets (2 and 4% NaCl) on the development of hypertension and obesity, oxidative stress, and renal function. Both 2 and 4% NaCl induced an early increase in systolic blood pressure of OP but not OR or C rats. High-salt intake induced an increase in the size and reduction in number of adipocytes, concomitant to a twofold increase in circulating leptin in OP rats. Aortic superoxide generation indicated a 2.8-fold increase in the OP high-salt vs. normal-salt groups, whereas urine isoprostanes were not significantly increased. Also, hydroxynonenal protein adducts in the kidney were highly increased in OP rats on 2 and 4% NaCl, indicating oxidative stress in the renal tissue. Urine albumin was increased threefold in the OP on 2% NaCl and fourfold in the same group on 4% NaCl vs. 0.8% NaCl. Kidney histology indicated a higher degree of glomerulosclerosis in OP rats on high-salt diets. In summary, high-salt diet accelerated the development but did not increase the severity of hypertension; high salt increased oxidative stress in the vasculature and kidney and induced kidney glomerulosclerosis and microalbuminuria. Also, the OP rats on high salt displayed adipocyte hypertrophy and increased leptin production.

Adaptation of resistance arteries to increases in pressure.

During the development of hypertension, hypertrophy of smooth muscle cells and deposition of extracellular matrix thicken the walls of large arteries without reducing the size of the lumen. The small arteries and arterioles remodel inwardly through a eutrophic process of rearrangement of the same smooth muscle cells around a smaller lumen. Pressure, through an increase in circumferential wall stress, can account for both hypertrophy and inward, eutrophic remodeling. The small arteries constrict during an elevation of pressure, thus restoring wall stress toward control levels. The large arteries have little vasoactivity and respond to the increase in wall stress by initiating a growth process. Mechanotransduction of the pressure stimulus to a growth response is being studied in small mesenteric arteries. Raising the pressure from 90 to 140 mmHg initiates a signaling process starting with phosphorylation of Src within 1 minute. This is followed by phosphorylation of Erk 1/2 peaking at 5 minutes and expression of c-fos mRNA within 30 minutes. Gene expression correlates with wall stress and is thus inhibited by a myogenic response. Maintained vasoconstriction in an isolated arteriole results in inward, eutrophic remodeling within 4 days. Thus, the current data support the hypothesis that wall thickness is determined by circumferential wall stress, and lumen size is determined by vascular tone.

Src autophosphorylation is an early event in pressure-mediated signaling pathways in isolated resistance arteries.

Elevated blood pressure is associated with varying degrees of arterial growth and remodeling. The mechanisms by which mechanical stress is converted into cellular alteration have yet to be fully elucidated. Our laboratory has demonstrated that Src tyrosine kinases and the extracellular signal-regulated kinase subtype of the mitogen-activated protein kinase family mediate pressure-induced c-fos expression in rat mesenteric arteries. Others have reported involvement of integrin and growth factor receptor signaling pathways. Our goal was to determine the role of Src, focal adhesion kinase (FAK), and platelet-derived growth factor (PDGF) receptor signaling in the upstream initiation of these events. Pairs of rat mesenteric arteries were pressurized to 90 mm Hg (control), and then one was raised to 140 mm Hg for 1, 3, or 5 minutes. Western blotting revealed that Src-pY(418) was elevated 2.4-fold over control values at 1 minute and 2.8-fold at 3 minutes and returned to control at 5 minutes. Significant FAK-Y(397) phosphorylation was observed only after 3 and 5 minutes of pressure stimulus and was blocked entirely by Src inhibition. Src-pY(215) activity (associated with PDGF receptor activation) does not seem to be involved at any of the time points tested. These data demonstrate that Src-Y(418) autophosphorylation is an early event in pressure mechanotransduction and leads to activation of FAK-Y(397). This finding suggests that Src may be the messenger that initiates and propagates the cellular growth response to pressure stimulus, and FAK is one of its downstream targets. Src phosphorylation due to PDGF receptor activation does not seem to be involved in the initial response.