Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

RATIONALE: Angiotensin-converting enzyme (ACE)2 opposes the actions of angiotensin (Ang) II by degrading it to Ang 1-7. OBJECTIVE: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. METHODS AND RESULTS: C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-α. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10(-7) mol/L). CONCLUSIONS: Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

Eplerenone does not attenuate diabetes-associated atherosclerosis.

BACKGROUND: It has been suggested that aldosterone, with its known pro-inflammatory and profibrotic actions, may play a key role in the development and progression of atherosclerosis. METHOD: In this study, the ability of aldosterone antagonism to reduce atherosclerosis in experimental diabetes was assessed. Diabetes was induced in ApoE knockout mice with streptozotocin, and the mice were treated with the specific aldosterone antagonist, eplerenone, in their feed over 20 weeks (approximately 200 mg/kg per day). RESULT: En face analysis revealed that eplerenone treatment was unable to attenuate atherosclerosis as assessed by percentage lesion area quantitation in the aortae of these mice compared with untreated diabetic mice (diabetic, 10.7 +/- 1.1; diabetic + eplerenone, 8.8 +/- 1.2%). In contrast, we observed a significant, more than 50% decrease in percentage of plaque area in the nondiabetic control groups. Despite this lack of effect in the diabetic mice, eplerenone treatment was associated with reduced cytosolic superoxide production. However, aortic transcript levels of key molecules implicated in diabetes-associated atherogenesis, such as monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1, were not significantly attenuated by eplerenone. CONCLUSION: These findings suggest that eplerenone treatment may not be as antiatherosclerotic in the diabetic context.

Receptor for advanced glycation end products (RAGE) deficiency attenuates the development of atherosclerosis in diabetes.

OBJECTIVE: Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE(-/-) model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS: ApoE(-/-) and RAGE(-/-)/apoE(-/-) double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS: Although diabetic apoE(-/-) mice showed increased plaque accumulation (14.9 +/- 1.7%), diabetic RAGE(-/-)/apoE(-/-) mice had significantly reduced atherosclerotic plaque area (4.9 +/- 0.4%) to levels not significantly different from control apoE(-/-) mice (4.3 +/- 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-kappaB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE(-/-) mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE(-/-)/apoE(-/-) mice. CONCLUSIONS: This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

Advances in the renin-angiotensin-aldosterone system: relevance to diabetic nephropathy.

Hypertension is now recognized as a key contributory factor to the development and progression of kidney disease in both type 1 and type 2 diabetes. The renin angiotensin system (RAS) and its effector molecule angiotensin II, in particular, have a range of hemodynamic and nonhemodynamic effects that contribute not only to the development of hypertension, but also to renal disease. As a result, therapeutic inhibition of the RAS with angiotensin-converting enzyme inhibitors and/or selective angiotensin II type 1 receptor blockers has been proposed as a key strategy for reducing kidney damage beyond the expected effects one would observe with blood pressure reduction per se. Although the relationship between the RAS and the progression of diabetic renal disease has been known for many decades, recent advances have revealed a more complex paradigm with the discovery of a number of new components. Thus, further understanding of these new components of the renin angiotensin aldosterone system (RAAS), such as the angiotensin type 2 receptor subtype, angiotensin converting enzyme 2, and the recently cloned renin receptor, is likely to have therapeutic implications for disorders such as diabetic nephropathy, where interruption of the RAAS is widely used.

Early vasodilator response to anodal current application in human is not impaired by cyclooxygenase-2 blockade.

It is generally acknowledged that cutaneous vasodilatation in response to monopolar galvanic current application would result from an axon reflex in primary afferent fibers and the neurogenic inflammation resulting from neuropeptide release. Previous studies suggested participation of prostaglandin (PG) in anodal current-induced cutaneous vasodilatation. Thus the inducible cyclooxygenase (COX) isoform (COX-2), assumed to play a key role in inflammation, should be involved in the synthesis of the PG that is released. Skin blood flow (SkBF) variations induced by 5 min of 0.1-mA monopolar anodal current application were evaluated with laser-Doppler flowmetry on the forearm of healthy volunteers treated with indomethacin (COX-1 and COX-2 inhibitor), celecoxib (COX-2 inhibitor), or placebo. SkBF was indexed as cutaneous vascular conductance (CVC), expressed as percentage of heat-induced maximal CVC (%MVC). Urinalyses were performed to test celecoxib treatment efficiency. No difference was found in CVC values at rest: 14.3 +/- 4.0, 11.9 +/- 3.2, and 10.9 +/- 2.0% MVC after indomethacin, celecoxib, and placebo treatment, respectively. At 10 min after the onset of anodal current application, CVC values were 22.2 +/- 4.9% MVC (not significantly different from rest) with indomethacin, 85.7 +/- 15.3% MVC (P < 0.001 vs. rest) with celecoxib, and 70.4 +/- 13.1% MVC (P < 0.001 vs. rest) with placebo. Celecoxib significantly depressed the urinary prostacyclin metabolite 6-keto-PGF(1alpha) (P < 0.05 vs. placebo). Indomethacin, but not celecoxib, significantly inhibited the anodal current-induced vasodilatation. Thus, although they are assumed to result from an axon reflex in primary afferent fibers and neurogenic inflammation, these results suggest that the early anodal current-induced vasodilatation is mainly dependent on COX-1-induced PG synthesis.

Spectral components of laser Doppler flowmetry signals recorded in healthy and type 1 diabetic subjects at rest and during a local and progressive cutaneous pressure application: scalogram analyses.

A significant transient increase in laser Doppler flowmetry (LDF) signals is observed in response to a local and progressive cutaneous pressure application in healthy subjects. This reflex may be impaired in diabetic patients. The work presents a signal processing providing the clarification of this phenomenon. Scalogram analyses of LDF signals recorded at rest and during a local and progressive cutaneous pressure application are performed on healthy and type 1 diabetic subjects. Three frequency bands, corresponding to myogenic, neurogenic and endothelial related metabolic activities, are studied. The results show that, at rest, the scalogram energy of each frequency band is significantly lower for diabetic patients than for healthy subjects, but the scalogram relative energies do not show any statistical difference between the two groups. Moreover, the neurogenic and endothelial related metabolic activities are significantly higher during the progressive pressure than at rest, in healthy and diabetic subjects. However, the relative contribution of the endothelial related metabolic activity is significantly higher during the progressive pressure than at rest, in the interval 200-400 s following the beginning of the pressure application, but only for healthy subjects. These results may improve knowledge on cutaneous microvascular responses to injuries or local pressures initiating diabetic complications.

Early endothelial dysfunction severely impairs skin blood flow response to local pressure application in streptozotocin-induced diabetic mice.

Pressure-induced vasodilation (PIV) is a mechanism whereby skin blood flow increases in response to progressive locally applied pressure. Skin blood flow in response to applied pressure decreased early in diabetic patients as a result of vascular and/or neural impairment. This study was designed to determine the effect of vascular changes on PIV in 1-week streptozotocin-induced diabetic mice. We assessed cutaneous microvascular response to local increasing pressure application measured by laser Doppler flowmetry (LDF) and endothelium-dependent and -independent vasodilation by iontophoretic delivery of acetylcholine and sodium nitroprusside and sciatic motor nerve conduction velocity and morphometry. In control mice, LDF increased 34% from baseline to 0.2 kPa external pressure, showing PIV response. In contrast, diabetic mice had no LDF increase in response to progressive external pressure. Moreover, after iontophoretic delivery of acetylcholine, endothelium-dependent vasodilation was largely attenuated in diabetic mice (25%) compared with control mice (81%), whereas vasodilation to sodium nitroprusside was not different between groups. Nerve function as assessed by sciatic nerve conduction velocity and morphometry did not differ between groups. These findings suggest that endothelial impairment is sufficient to severely alter PIV response, which seems to be highly sensitive to endothelial nitric oxide levels. PIV suppression could favor diabetes complications such as diabetic foot ulcers.

Time-frequency analysis of laser Doppler flowmetry signals recorded in response to a progressive pressure applied locally on anaesthetized healthy rats.

The laser Doppler flowmetry technique has recently been used to report a significant transient increase of the cutaneous blood flow signal, in response to a local non-noxious pressure applied progressively on the skin of both healthy humans and rats. This phenomenon is not entirely understood yet. In the present work, a time-frequency analysis is applied to signals recorded on anaesthetized healthy rats, at rest and during a cutaneous pressure-induced vasodilation (PIV). The comparison, at rest and during PIV, of the scalogram relative energies and scalogram relative amplitudes in five bands, corresponding to five characteristic frequencies, shows an increased contribution for the endothelial related metabolic activity in PIV signals, till 400 s after the beginning of the progressive pressure application. The other subsystems (heart, respiration, myogenic and neurogenic activities) contribute relatively less during PIV than at rest. The differences are statistically significant for all the relative activities in the interval 0-200 s following the beginning of the pressure. These results and others obtained on patients, such as diabetics, could increase the understanding of some cutaneous pathologies involved in various neurological diseases and in the pathophysiology of decubitus ulcers.

Low skin temperature impairs the cutaneous vasodilator response to local progressive pressure strain.

A pressure-induced vasodilation (PIV) was recently reported as a putative protective response in human skin. Therefore, we examined the influence of skin temperatures on cutaneous blood flow responses to local progressive pressure strain. Ten healthy volunteers were studied at different ambient temperatures leading to low (29.0 +/- 0.3 degrees C), intermediate (32.6 +/- 0.1 degrees C), high (33.9 +/- 0.1 degrees C) and very high (36.0 +/- 0.1 degrees C) skin temperatures. We measured cutaneous blood flow using laser Doppler flowmetry on the foot in response to a local progressive pressure increase of 5.0 mm Hg min(-1). Progressive pressure strain led to an almost linear cutaneous laser Doppler flow decrease at both low and intermediate skin temperatures (-40.1 +/- 6.6% and -31.2 +/- 6.5% from baseline at 30 +/- 1.25 mm Hg), whereas at both high and very high skin temperatures, subjects responded with a transient cutaneous vasodilation (+33.6 +/- 10.6% and +50.6 +/- 15.4% from baseline at 30 +/- 1.25 mm Hg). These findings suggest that high skin temperatures are required for the PIV to develop.

Impaired pressure-induced vasodilation at the foot in young adults with type 1 diabetes.

Vascular and neurological mechanisms are both likely to be involved in foot ulcer. We recently reported a pressure-induced vasodilation (PIV), relying on unmyelinated afferent excitation. We previously found that cutaneous blood flow in response to locally applied pressure might be impaired in diabetic patients because of the combined effects of low cutaneous temperature and alterations in microcirculatory function. Therefore, we aimed to analyze whether, at a relatively high cutaneous temperature, PIV is present in type 1 diabetes and to assess endothelial-dependent vasodilation and endothelium-independent vasodilation. We measured cutaneous blood flow using laser Doppler flowmetry on the head of the first metatarsus in response to applied pressure at 5.0 mmHg/min in warm conditions (29.5 +/- 0.2 degrees C). Responses to iontophoresis of acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent) were measured using laser Doppler flowmetry in the forearm. The data indicate that PIV exists at the foot level in normal subjects, whereas it was not found in diabetic patients. In diabetic patients, the nonendothelial-mediated response to sodium nitroprusside was preserved, whereas the endothelial-mediated response to acetylcholine was impaired. These findings might be relevant to the high prevalence of foot ulcer that occurs in diabetic patients.

Wavelet de-noising of laser Doppler reactive hyperemia signals to diagnose peripheral arterial occlusive diseases.

In order to improve peripheral arterial occlusive diseases (PAOD) diagnoses, five de-noising algorithms based on a multiresolution analysis computed with wavelets are applied on reactive hyperemia signals obtained with the laser Doppler flowmetry technique. Results are presented on recordings acquired on patients suffering from PAOD and on healthy subjects.