Didier Heudes

Evidence for in vivo carotid and femoral wall thickening in human hypertension. Groupe de Prévention Cardio-vasculaire en Médecine du Travail.

Little is known of the in vivo structural changes of large arteries in uncomplicated hypertension. Therefore, we measured the intima-media thickness and lumen diameter of common carotid and femoral arteries by a computerized ultrasonographic technique in 25 normotensive and 25 never treated hypertensive men of similar age (from 25 to 72 years). The intraobserver variability of carotid and femoral wall thicknesses was 4.3% and 5.6%, respectively. Moreover, an in vitro study of 13 human arterial segments removed at autopsy demonstrated a strong correlation (r = .989, P < .001) between computerized ultrasonic and histological intima-media thickness measurements. Compared with control subjects, hypertensive patients had similar arterial diameters but higher carotid and femoral intima-media thicknesses (P < .001) as well as higher ratios of carotid and femoral intima-media thickness to lumen (P < .001, P < .01). The carotid thickness was correlated with age in control subjects (r = .48, P < .05) but not in hypertensive patients. The femoral thickness was correlated with age both in control subjects (r = .55, P < .01) and in hypertensive patients (r = .46, P < .05). Thus, carotid and femoral arterial walls of hypertensive patients were thickened. This thickening was not due to age, although aging also thickened both vessels in control subjects and the femoral artery only in hypertensive patients. Such a wall thickening associated with a normal diameter provides direct evidence of vascular growth and represents a new target to monitor noninvasively in vivo for large artery changes in human hypertension.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to βadrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein–kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein–kinin system could be involved in the development or progression of cardiovascular diseases.

Genetically increased angiotensin I-converting enzyme level and renal complications in the diabetic mouse

Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseases and has a marked genetic component. A common variant (D allele) of the angiotensin Iconverting enzyme (ACE) gene, determining higher enzyme levels, has been associated with diabetic nephropathy. To address causality underlying this association, we induced diabetes in mice having one, two, or three copies of the gene, normal blood pressure, and an enzyme level range (65–162% of wild type) comparable to that seen in humans. Twelve weeks later, the three-copy diabetic mice had increased blood pressures and overt proteinuria. Proteinuria was correlated to plasma ACE level in the three-copy diabetic mice. Thus, a modest genetic increase in ACE levels is sufficient to cause nephropathy in diabetic mice.

Left Ventricular Fibrosis in Renovascular Hypertensive Rats Effect of Losartan and Spironolactone

Introduction: Myocardial fibrosis associated with arterial hypertension modifies myocardial function and structure. Using spironolactone (spiro), an aldosterone antagonist, and losartan (DuP 753), an angiotensin II antagonist, we attempted to elucidate the role played by these hormones in the establishment of myocardial fibrosis in the renovascular Goldblatt model (two-kidney, one clip). Methods: Sixty-three male Wistar rats were used. Ten clipped rats and eleven age-matched controls were killed 1 month after the application of a renal clip. Eight other clipped rats received 20 mg/kg per day spiro per os, twelve received 20 mg/kg per day losartan and nine received placebo; all were killed 4 months after clipping, together with 13 controls. The left ventricles were removed and weighed. The total collagen content, including pericoronary and microscar coflagens, was estimated macroscopically (MacroColl) on paraffin sections stained with Sinus red. Otherwise, the fibrosis was estimated with polarization microscopy in which type I collagen (coll I) is orange-coloured and type III collagen (coll III) is green. Thus, using appropriate band-pass filters and computer-assisted morphometry, it was possible to quantitate each collagen. Results: One month after clipping, and throughout the experiment, regardless of treatment, systolic blood pressure (SBP) and left ventricular hypertrophy (LV/BW) were significantly increased in the clipped groups. The MacroColl was significantly increased in the clipped groups. Losartan, unlike spiro, prevented this increase. The interstitial coll I and coll III densities (IntDens) were not increased in the untreated clipped rats. The coll III IntDens was lower in the spiro group than in all age-matched groups. The pericoronary coll III content weighed by the surface area of the artery (PeriColl III) was increased significantly from the first to the fourth month after clipping in all clipped rats except those treated with spiro. The increase in PeriColl I 4 months after clipping was prevented by losartan but not by spiro treatment (Table 1). Conclusions: Together with hypertension, aldosterone and angiotensin II play an important role on myocardial fibrosis, inducing respectively coll III and coll I synthesis, mainly around the coronary artery. Although the coll III precedes the coll I synthesis, coll III synthesis inhibition by spiro treatment did not affect coll I synthesis. This suggests that these two types of collagen have different metabolisms.

Correlation between clinical and histologic patterns of degenerative mitral valve insufficiency: a histomorphometric study of 130 excised segments.

The objectives of this study were to examine quantitatively the histological changes in incompetent degenerative mitral valves obtained at surgery for mitral valve repair, and to determine whether Barlows disease (BD) and fibroelastic deficiency (FED) can be distinguished by histology. The billowing mitral leaflet syndrome (or Barlows disease) and FED can be distinguished on the basis of clinical patterns and gross features, but their histologic patterns have not been described. One hundred thirty patients were studied. Thirty-nine (24 males) had BD; 44 (38 males) FED; 15 (7 males) Marfans syndrome (MS); and 32 patients (25 males) a non-determined degenerative disease. Histological changes of the resected segment of the valve were quantitatively evaluated using scores of severity. A discriminant analysis was performed. The groups defined by the computer were checked for concordance with groups defined by the surgeon. Collagen alterations were found the most severe in MS patients. BD and MS had the most myxoid infiltration. MS and FED patients had the most elastic fiber alterations. No BD in males and only one in females were misclassified by the discriminant procedure into the FED group. Overall, the percentages of correct matchings were 54% in males and 62% in females. When the age of patients and the size of ring were added to histology to determine whether this additional information provided more discrimination, the percentages of correct matchings reached 90% in males and 100% in females. BD and FED are two fairly distinct entities, which can be distinguished by quantitative histology, whereas only modest differences were found in qualitative histology.

Peroxisome Proliferator-Activated Receptor β/δ Exerts a Strong Protection from Ischemic Acute Renal Failure

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Noninvasive Measurement of Medium-Sized Artery Intima-Media Thickness in Humans: In vitro Validation

Recent research in ultrasound technology has led to the development of a high-resolution echo-tracking device. The present study was performed to evaluate the accuracy in the measurement of human radial artery intima-media thickness with this new device. We determined the correlation between histological and ultrasonic measurements of intima-media thickness in 15 radial artery segments obtained from the distal end of the wrist-elbow harvest for coronary bypass grafting in patients with coronary heart disease. For arterial intima-media thickness, a positive correlation was observed between ultrasonic and histological measurements (r = 0.618; p < 0.014), and the difference between ultrasound and histology measurements was 41 +/- 66 microns, with the higher measurements found by the ultrasonic device. In a subgroup of 11 patients, we determined the correlation between in vivo ultrasonic measurements of radial artery intima-media thickness at the preoperative stage and in vitro ultrasonic measurements of intima-media thickness obtained postoperatively in the same arterial segments. Internal diameter was larger in vivo than in vitro, and intima-media thickness was smaller in vivo than in vitro. The cross-sectional area of the arterial wall was calculated from internal diameter and intima-media thickness. In vitro wall cross-sectional area was correlated with in vivo wall cross-sectional area (r = 0.929; p < 0.0001). Repeatability of in vivo intima-media thickness measurements was investigated in 10 subjects through the calculation of the repeatability coefficient as defined by the British Standards Institution.(ABSTRACT TRUNCATED AT 250 WORDS)

Respective role of humoral factors and blood pressure in cardiac remodeling of DOCA hypertensive rats

OBJECTIVES Recent studies have shown that beside elevated arterial blood pressure, humoral factors such as angiotensin II, aldosterone, endothelin or bradykinin might play a role in the cardiac hypertrophy and fibrosis secondary to hypertension. In addition, it seems that perivascular fibrosis and interstitial fibrosis are controlled by independent mechanisms. Therefore, the goal of our study was to evaluate the respective role of the increased arterial pressure and of humoral factors on cardiac remodeling in an experimental hypertension model. METHODS Uninephrectomized rats received DOCA, a high salt diet, and when hypertension was installed, they were treated for 6 weeks with either a long-acting calcium antagonist, mibefradil (30 mg/kg day-1), an ACE inhibitor, enalapril (3 mg/kg day-1), or a mixed ETA-ETB endothelin receptor antagonist, bosentan (100 mg/kg day-1). A group of hypertensive rats was left untreated and a sham-operated group of normotensive rats was used for control. At the end of treatment, maximal coronary blood flow was measured in isolated perfused hearts. Cardiac hypertrophy and interstitial as well as perivascular fibrosis were evaluated by quantitative morphometry. RESULTS DOCA-salt hypertensive rats exhibited a marked cardiac hypertrophy associated with a decrease of maximal coronary blood flow and interstitial and perivascular fibrosis. The calcium antagonist nearly normalized arterial pressure and suppressed all these changes. Enalapril had no effect on arterial pressure and perivascular fibrosis but decreased subendocardial fibrosis. Bosentan had a very small effect on arterial pressure but decreased cardiac hypertrophy and both perivascular and subendocardial fibrosis. CONCLUSIONS We conclude that in DOCA salt hypertension, humoral factors such as endothelin may play a role beside high blood pressure in cardiac remodeling. In addition, the different components of this remodeling (decrease of vascular reserve, cardiac hypertrophy and cardiac fibrosis) are controlled independently.

Inflammatory cells and myocardial fibrosis: spatial and temporal distribution in renovascular hypertensive rats

OBJECTIVE The fibroblasts producing collagen are co-localized with inflammatory cells in myocardial fibrosis areas of spontaneously hypertensive rats, suggesting that collagen overproduction in this model may be modulated by inflammatory cells. The present study extends these observations to the Goldblatt model of hypertension in which the renin-angiotensin system is activated. METHODS Inflammatory cells were identified with monoclonal antibodies directed against macrophages (ED1+), T helper (CD4+) and cytotoxic lymphocytes (CD8+), and MHC class II-expressing cells (Ia+). The alkaline phosphatase-anti-alkaline phosphatase (APAAP) immuno-staining technique was used. A new computer-assisted morphometric method was utilized to quantify the inflammatory infiltrate in each cardiac compartment with polarized-light microscopy. Cells responsible for the collagen synthesis were identified by in situ hybridization. The collagen content was estimated by morphometry on left ventricle sections stained with Sirius red, and by biochemical quantification of the hydroxyproline concentration. RESULTS Computer-assisted morphometry under polarized light was well suited to quantify inflammatory cells labeled by the APAAP technique. Inflammatory cells were co-localized with collagen-synthesizing fibroblasts. The main inflammatory cells were CD4+ lymphocytes > Ia+ > ED1+ > CD8+ cells. These cell densities were increased in hypertensive rats in all cardiac areas compared to control rats except for IA+ cells which were concentrated in microscars. Macrophage density was correlated with plasma renin activity. The inflammatory cell density which best correlated with fibrosis was macrophage density, and which best correlated with systolic blood pressure was macrophage and T helper lymphocyte densities. CONCLUSIONS One can speculate that the correlation between macrophage density and blood pressure as well as with plasma renin activity may indicate that angiotensins and/or elevation of blood pressure could participate in the initial signalling which may mobilize inflammatory cells. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors or cytokines which act upon fibroblasts.

Endothelin Antagonism in End-Organ Damage of Spontaneously Hypertensive Rats: Comparison With Angiotensin-Converting Enzyme Inhibition and Calcium Antagonism

High blood pressure results in cardiac hypertrophy and fibrosis, increased thickness and stiffness of large artery walls, and decreased renal function. The objective of our study was to assess the role of endothelin, angiotensin II, and high blood pressure in the end-organ damage observed in spontaneously hypertensive rats (SHR). For this purpose, SHR were treated for 10 weeks with either a mixed endothelin-A and endothelin-B receptor antagonist, bosentan (100 mg/kg per day), an angiotensin-converting enzyme inhibitor, enalapril (10 mg/kg per day), or a long-acting calcium antagonist, mibefradil (20 mg/kg per day). A group of SHR was left untreated, and a group of normotensive Wistar rats was used as control. At the end of treatment, maximal coronary blood flow was measured in isolated perfused hearts. Cardiac hypertrophy and fibrosis, aortic medial thickness, and extracellular matrix content were evaluated by quantitative morphometry. Proteinuria and urea and creatinine clearances were measured, and renal histopathology was assessed. SHR exhibited cardiac hypertrophy, perivascular fibrosis, and decreased maximal coronary blood flow. Aortic medial thickness was increased, whereas elastin density was decreased. Finally, SHR showed decreased urinary excretion and decreased urea and creatinine clearances. No renal histological lesions were observed. Although bosentan did not affect blood pressure, it normalized renal function and slightly decreased left ventricular hypertrophy and fibrosis. Enalapril and mibefradil were both effective in significantly decreasing blood pressure, left ventricular hypertrophy, and aortic medial thickness and improving coronary blood flow, but in contrast to bosentan, they did not improve creatinine clearance. We conclude that in SHR, high blood pressure plays a major role in end-organ damage and that endothelin may partly mediate renal dysfunction and cardiac remodeling independently of a direct hemodynamic effect.