Impact of metabolic indices on central artery stiffness: independent association of insulin resistance and glucose with aortic pulse wave velocity.

AIMS/HYPOTHESIS: Non-invasive measures of aortic stiffness reflect vascular senescence and predict outcome in diabetes. Glucose-mediated elastic artery sclerosis may play an integral role in the development of macrovascular complications. We used carotid-femoral pulse wave velocity ((cf)PWV) to quantify independent associations of fasting glucose, post-challenge glucose and derived insulin resistance (HOMA-IR) with aortic stiffness. METHODS: (cf)PWV was measured using a 4 MHz continuous wave Doppler ultrasound probe within groups with newly identified age- and sex-matched normal glucose metabolism (NGM), impaired glucose regulation (IGR) and diabetes mellitus populations (n = 570, mean age 59.1, 56% male). RESULTS: After multivariate adjustment, IGR and diabetes were associated with significant aortic stiffening compared with NGM (adjusted (cf)PWV+/-SE: NGM, 9.15 +/- 0.12 m/s; IGR 9.76 +/- 0.11 m/s, p < 0.001; diabetes, 9.89 +/- 0.12 m/s, p < 0.001). IGR stratification indicated that impaired fasting glucose (IFG; 9.71 +/- 0.12 m/s) and post-challenge (impaired glucose tolerance; 9.82 +/- 0.24 m/s) categories had similar (cf)PWV (p = 0.83). Modelled predictors of (cf)PWV were used to assess independent metabolic associations with arterial stiffness. Fasting glucose concentration (beta = 0.10; 95% CI 0.05, 0.18; p = 0.003), 2 h post-challenge glucose (beta = 0.14; 95% CI 0.02, 0.23; p < 0.001) and HOMA-IR (beta = 0.20, 95% CI 0.05, 0.53; p < 0.001) were independently related to (cf)PWV after adjustment for age, sex, mean arterial pressure, heart rate, body mass index, renal function and antihypertensive medication. CONCLUSIONS/INTERPRETATION: IGR characterised by fasting or post-challenge hyperglycaemia is associated with significant vascular stiffening. Post-challenge glucose and HOMA-IR are the most powerful metabolic predictors of arterial stiffness, implying hyperglycaemic excursion and insulin resistance play important roles in the pathogenesis of arteriosclerosis.

Telomere attrition and accumulation of senescent cells in cultured human endothelial cells.

The human umbilical vein endothelial cell (HUVEC) is an important model of the human endothelium that is widely used in vascular research. HUVECs and the adult endothelium share many characteristics including progression into senescence as the cells age. Despite this, the shortening of telomeres and its relationship to the progression into senescence are poorly defined in HUVECs. In this study of several HUVEC lines we show notable consistency in their growth curves. There is a steady decline in the growth rate of HUVECs grown continually in culture and we estimate complete cessation of growth after approximately 70 population doublings. The HUVECs lose telomeric DNA at a consistent rate of 90 base pairs/population doubling and show a progressive accumulation of shortened telomeres (below 5 kilobases). This telomeric loss correlates with the accumulation of senescent HUVECs in culture as assessed by staining for beta-galactosidase activity at pH 6. Although the telomere length of a large population of cells is a relatively crude measure, we suggest that in HUVECs a mean telomere length (as measured by terminal restriction fragment length) of 5 kilobases is associated with entry into senescence. These data demonstrate the strong relationship between telomere attrition and cell senescence in HUVECs. They suggest that DNA damage and subsequent telomere attrition are likely to be key mechanisms driving the development of endothelial senescence in the pathogenesis of vascular disease.

The expression of renin-binding protein and renin in the kidneys of rats with two-kidney one-clip hypertension.

OBJECTIVE: To test the hypothesis that renin-binding protein (RnBP) is involved in modulating the intracellular processing or release of renin, we examined the expression of RnBP in clipped and contralateral kidneys of rats with two-kidney one-clip hypertension, and in left and right kidneys from sham-operated control rats. DESIGN AND METHODS: Kidneys from rats with two-kidney one-clip hypertension and from control rats were either snap-frozen for extraction of mRNA or fixed for in-situ hybridization and immunochemistry. Reverse-transcription polymerase chain reaction on renal mRNA was performed using primers for renin, RnBP, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and angiotensin-converting enzyme (ACE). In addition, renal total RNA was analysed by Northern blotting for RnBP, GAPDH and angiotensin II type 1A (AT1A) receptor mRNA, and the intensity of the bands was measured by laser densitometry. In situ hybridization for renin mRNA was carried out using digoxygenin-labelled antisense oligonucleotides and for RnBP using labelled antisense oligonucleotides and an antisense riboprobe. Controls included sections treated with RNase and sections stained with sense oligonucleotides. RESULTS: The level of expression of mRNA for RnBP is similar in clipped and contralateral kidneys of renal hypertensive rats; in contrast, renin mRNA expression is upregulated in the clipped kidney. Renin-binding protein is expressed mainly in renal tubules and collecting ducts unlike renin, which is expressed in the glomerular afferent arteriole. We did not detect lateralization of expression for ACE or the AT1A receptor between clipped and contralateral kidneys. CONCLUSION: Renin-binding protein expression is unchanged between clipped and contralateral kidneys. Therefore, a physiological stimulus that upregulates renin gene expression in clipped kidneys does not affect RnBP expression. The main sites of RnBP expression are the renal tubules and collecting ducts; in contast renin is expressed at the glomerular pole. The results show that RnBP is not colocalized or coregulated with renin in this model of hypertension.

Evidence against potassium as an endothelium-derived hyperpolarizing factor in rat mesenteric small arteries.

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.

A non-traumatic approach to ventral subarachnoid space near circle of Willis.

A non-traumatic approach to the subarachnoid space near the circle of Willis is demonstrated in this study. The approach is made between the olfactory bulb and frontal lobe. Following perfusion with Evans blue dye no leakage was observed into the parenchyma indicating no tissue damage had occurred. No gross bleeding or cerebral vasospasm was visible in the middle meningeal artery (dural), the surface pial blood vessels and the vessels of the circle of Willis. In conclusion, this technique can be successfully used for studies on experimental subarachnoid hemorrhage simulated on the ventral aspect of the brain near the circle of Willis without causing technique-associated cerebrovascular and parenchymal injury.

Morphometric evaluation of brain infarcts in rats and gerbils.

The Levine rat preparation, the gerbil stroke model, and appropriate control animals were used to determine if the 2,3,5-triphenyltetrazolium chloride (TTC) would selectively identify noninfarcted versus infarcted cerebral tissue. The TTC is frequently used to quantify infarcted myocardial tissue and has been shown to have great specificity, reproducibility, and efficacy. The TTC produces a red product upon reaction with the respiratory enzymes (dehydrogenases) present in non-infarcted tissues. Irreversibly damaged tissues, lacking dehydrogenases, do not form red reaction products. Six gerbil brains and seven rat brains were incubated with the TTC, and the unreacted areas were macroscopically identified. The brains were fixed and sectioned for routine hematoxylin and eosin staining to determine the specificity of the TTC. The TTC was found to react selectively only with non-infarcted cerebral tissue. The gross brain sections were evaluated by macroscopic morphometric analysis, and the unreacted area was always ipsilateral to ligation and correlated with histologic identification of infarct. The brains from neurologically intact animals demonstrated neither macroscopic nor histological evidence of infarction. This technique allows macroscopic quantification of infarct size by planimetry. The average area of infarct for the neurologically impaired rats was 34.7% and it was 31.4% for the impaired gerbils. The percentage of surface area of each infarcted slice was found to correlate with the severity of the neurologic deficit. We conclude that TTC staining is effective for macroscopically delineating cerebral infarcts in rats and gerbils, thus permitting quantification of infarct size.

A correlation between multiple unit activity in the hypothalamus and electrocardiographic changes during a subarachnoid hemorrhage.

A sudden onset and short latency of cardiovascular responses that may follow a subarachnoid hemorrhage (SAH) in the vicinity of the circle of Willis implicate neural mechanisms. To investigate this, multi unit activity (MUA) was recorded from the posterolateral hypothalamus and electrocardiogram was recorded from lead II before and during SAH in rats. A temporal correlation between MUA, heart rate and rhythm changes was observed. Following SAH, transient depression of MUA (approximately 5 s or less) did not affect heart rate; a more sustained suppression preceded bradycardia and arrhythmias. Bradycardia, premature atrial contractions and premature ventricular contractions occurred under two conditions: when MUA was suppressed; and during bursts in MUA when interburst phases were suppressed. Further evidence that bradycardia may be the result of suppression of brain potentials was obtained by failure of bilateral vagotomy, atropine and isoproterenol to reverse bradycardia generated after SAH under the ventral aspect of the brain in the vicinity of circle of Willis. However, bilateral vagotomy reversed bradycardia after SAH in the cisterna magna indicating an activation of central parasympathetic neurons. The results indicate that different neural mechanisms underlie bradycardia generated from the above mentioned two sites, namely, ventral brain and cisterna magna.

Central neural control of blood pressure and cardiac arrhythmias during subarachnoid hemorrhage in rats.

Sudden death may follow subarachnoid hemorrhage which indicates involvement of neural mechanisms connected with the cardiovascular system. Since various regions of the brain mediate blood pressure and heart rate changes, these parameters and heart rhythm could be affected due to a subarachnoid hemorrhage near the circle of Willis which surrounds the hypothalamus, the highest center for autonomic control. To investigate this in the control group, intracranial pressure, blood pressure, and electrocardiogram were measured before and after a simulated subarachnoid hemorrhage; blood pressure and electrocardiogram were measured following midcollicular lesions in the decerebrate group both before and after a subarachnoid hemorrhage. The results demonstrate that an increase in systemic arterial blood pressure and premature ventricular contractions (with respect to unlesioned group, p less than 0.04) are mediated by forebrain areas and require the integrity of neuroanatomical connections with structures that are caudal to the midbrain. Since bradycardia and other electrocardiographic abnormalities could still be produced after midcollicular lesioning it is suggested that they can be mediated via the brainstem only without involvement of more rostral areas and may occur due to increased intracranial pressure.

A small animal model for electrocardiographic abnormalities observed after an experimental subarachnoid hemorrhage.

The male Sprague-Dawley rat was successfully used to produce increases in arterial blood pressure and electrocardiographic abnormalities similar to those seen in patients after a subarachnoid hemorrhage. The model employs a less expensive, easily available laboratory animal than other existing models. Whole heparinized blood is introduced in the area of the circle of Willis to simulate a subarachnoid hemorrhage. This model ensures a constant body-blood volume and minimizes damage to brain tissue as no blood vessel has to be punctured. The volume of blood required to produce bradycardia and other electrocardiographic abnormalities can be controlled and calculated for each animal. The model produced a sudden onset and progression of cardiovascular symptoms. Blood apparently is specific for producing electrocardiographic abnormalities as compared to 0.9% saline, Dextran 40 and Gentran 75 when introduced in the area of the circle of Willis. This specificity was not maintained when blood and saline were introduced into the cisterna magna as electrocardiographic abnormalities were observed with both agents.