Élise Roussel

Cell biology of caveolae and caveolin

Originally described in the 1950s caveolae are morphologically identifiable as small omega-shaped plasma membrane invaginations present in most cell types. Caveolae are particularly abundant in adipocytes, fibroblasts, type 1 pneumocytes, endothelial and epithelial cells as well as in smooth and striated muscle cells. The first proposed function for caveolae was that of mediating the internalisation and transendothelial trafficking of solutes. Caveolae have been the object of intense research since the discovery of a biochemical marker protein, caveolin, in the early 1990s. Three genes encoding for caveolins have been characterised in mammals. Caveolins (18-24 kDa) are integral membrane proteins that constitute the major protein component of caveolar membrane in vivo. In addition to a structural role of caveolins in the formation of caveolae, caveolin protein interacts directly, and in a regulated manner, with a number of signalling molecules. We present here a general overview of the current knowledge on the structural role of caveolin in caveolae formation, and implication of caveolin in the control of cell signalling.

Effectiveness of β-Blockade in Experimental Chronic Aortic Regurgitation

Background—Past studies have suggested that the adrenergic system becomes abnormally activated in chronic volume overload, such as in severe aortic valve regurgitation (AR). However, the effectiveness of agents directed against this adrenergic activation has never been adequately tested in chronic AR. We therefore tested the effects of metoprolol treatment on the left ventricular (LV) function and remodeling in severe chronic AR in rats. Methods and Results—Severe AR was created in adult male Wistar rats by retrograde puncture of the aortic leaflets under echocardiographic guidance. Two weeks later, some animals received metoprolol treatment (25 mg/kg) orally for 24 weeks, and some were left untreated. LV dimensions, ejection fraction, and filling parameters were evaluated by echocardiography. Hearts were harvested at 1, 2, 14, and 180 days for the evaluation of hypertrophy, &bgr;-adrenergic receptor status, and extracellular matrix remodeling. We found that metoprolol treatment prevented LV dilatation and preserved the ejection fraction and filling parameters compared with untreated animals. Metoprolol increased the expression of &bgr;1-adrenoreceptor mRNA and reduced G protein receptor kinase 2 levels. Collagen I and III mRNA levels were reduced. Cardiac myocyte hypertrophy was also prevented. Conclusions—In our experimental model of severe AR, metoprolol treatment had a significant beneficial global effect on LV remodeling and function. These results suggest that the adrenergic system is important in the development of volume-overload cardiomyopathy in AR and that adrenergic-blocking agents may play a role in the treatment of this disease.

Role of caveolin-1 in etoposide resistance development in A549 lung cancer cells.

Caveolin 1 expression is down-regulated in various cancer cell lines. Interestingly, in several drug-resistant cancer cells, a strong induction of caveolin 1 expression has been reported suggesting a role for caveolin 1 in the acquisition and/or the maintenance of multidrug resistance phenotype. In addition, it was reported that p-glycoprotein localized to caveolin-rich membrane domains in these cells. In this study, we progressively exposed A549 lung adenocarcinoma cells to increasing doses of etoposide. Both R1 and R2 cell lines had greatly increased levels of p-glycoprotein expression while mrp expression levels were moderately increased but only R2 cells had raised caveolin levels compared to control A549 cells. Both caveolin-1 and p-glycoprotein co-localize in Triton-insoluble membrane domains in all our cell lines but only caveolins-1 was solubilized by the addition of octylglucoside at 4C suggesting that these two proteins are located in different membrane domains. Using an anti-caveolin-1 antibody, we did not succeed to immunoprecipitate p-glycoprotein. Interestingly, total cellular cholesterol (the major lipid component of caveolae and triton-insoluble domains) was greatly increased in both R1 and R2 cell lines compared to naïve A549 cells.

Benefits of long-term β-blockade in experimental chronic aortic regurgitation

The objective of this study was to assess the long-term effects of beta-blockade on survival and left ventricular (LV) remodeling in rats with aortic valve regurgitation (AR). The pharmacological management of chronic AR remains controversial. No drug has been definitively proven to delay the need for valve replacement or to affect morbidity and/or mortality. Our group has reported that the adrenergic system is activated in an animal model of AR and that adrenergic blockade may help maintain normal LV function. The effects of prolonged treatment with a beta-blocker are unknown. Forty Wistar rats with severe AR were divided into 2 groups of 20 animals each and treated with metoprolol (Met, 25 mg.kg(-1).day(-1)) or left untreated for 1 yr. LV remodeling was evaluated by echocardiography. Survival was assessed by Kaplan-Meir curves. Hearts were harvested for tissue analysis. All Met-treated animals were alive after 6 mo vs. 70% of untreated animals. After 1 yr, 60% of Met-treated animals were alive vs. 35% of untreated animals (P = 0.028). All deaths, except one, were sudden. There were no differences in LV ejection fraction (all >50%) or LV dimensions. LV mass tended to be lower in the Met-treated group. There was less subendocardial fibrosis in this group, as well as lower LV filling pressures (LV end-diastolic pressure). beta-Adrenergic receptor ratio (beta(1)/beta(2)) was improved. One year of treatment with Met was well tolerated. Met improved 1-yr survival, minimized LV hypertrophy, improved LV filling pressures, decreased LV subendocardial fibrosis, and helped restore the beta-adrenergic receptor ratio.

Moderate Exercise Training Improves Survival and Ventricular Remodeling in an Animal Model of Left Ventricular Volume Overload

Background—Exercise training has beneficial effects in patients with heart failure, although there is still no clear evidence that it may impact on their survival. There are no data regarding the effects of exercise in subjects with chronic left ventricular (LV) volume overload. Using a rat model of severe aortic valve regurgitation (AR), we studied the effects of long-term exercise training on survival, development of heart failure, and LV myocardial remodeling. Methods and Results—One hundred sixty male adult rats were divided in 3 groups: sham sedentary (n=40), AR sedentary (n=80), and AR trained (n=40). Training consisted in treadmill running for up to 30 minutes, 5 times per week for 9 months, at a maximal speed of 20 m/minute. All sham-operated animals survived the entire course of the protocol. After 9 months, 65% of trained animals were alive compared with 46% of sedentary ones (P=0.05). Ejection fractions remained in the normal range (all above 60%) and LV masses between AR groups were similar. There was significantly less LV fibrosis in the trained group and lower LV filling pressures and improved echocardiographic diastolic parameters. Heart rate variability was also improved by exercise. Conclusion—Our data show that moderate endurance training is safe, does not increase the rate of developing heart failure, and most importantly, improves survival in this animal model of chronic LV volume overload. Exercise improved LV diastolic function, heart rate variability, and reduced myocardial fibrosis.

Dobutamine stress echocardiography in healthy adult male rats

BackgroundDobutamine stress echocardiography is used to investigate a wide variety of heart diseases in humans. Dobutamine stress echocardiography has also been used in animal models of heart disease despite the facts that the normal response of healthy rat hearts to this type of pharmacological stress testing is unknown. This study was performed to assess this normal response.Methods15 normal adult male Wistar rats were evaluated. Increasing doses of dobutamine were infused intravenously under continuous imaging of the heart by a 12 MHz ultrasound probe.ResultsDobutamine stress echocardiography reduced gradually LV diastolic and systolic dimensions. Ejection fraction increased by a mean of +24% vs. baseline. Heart rate increased progressively without reaching a plateau. Changes in LV dimensions and ejection fraction reached a plateau after a mean of 4 minutes at a constant infusion rate.ConclusionDSE can be easily performed in rats. The normal response is an increase in heart rate and ejection fraction and a decrease in LV dimensions. A plateau in echocardiographic measurements is obtained after 4 minutes of a constant infusion rate in most animals.

Gene profiling of left ventricle eccentric hypertrophy in aortic regurgitation in rats: rationale for targeting the β-adrenergic and renin-angiotensin systems

Aortic valve regurgitation (AR) imposes a severe volume overload to the left ventricle (LV), which results in dilation, eccentric hypertrophy, and eventually loss of function. Little is known about the impact of AR on LV gene expression. We, therefore, conducted a gene expression profiling study in the LV of rats with acute and severe AR. We identified 64 genes that were specifically upregulated and 29 that were downregulated out of 21,910 genes after 2 wk. Of the upregulated genes, a good proportion was related to the extracellular matrix. We subsequently studied a subset of 19 genes by quantitative RT-PCR (qRT-PCR) to see if the modulation seen in the LV after 2 wk persisted in the chronic phase (after 6 and 12 mo) and found that it did persist. Knowing that the adrenergic and renin-angiotensin systems are overactivated in our animal model, we were interested to see if blocking those systems using metoprolol (25 mg.kg(-1).day(-1)) and captopril (100 mg.kg(-1).day(-1)) would alter the expression of some upregulated LV genes in AR rats after 6 mo. By qRT-PCR, we observed that upregulations of LV mRNA levels encoding for procollagens type I and III, fibronectin, atrial natriuretic peptide, transforming growth factor-beta(2), and connective tissue growth factor were totally or partially reversed by this treatment. These observations provide a molecular rationale for a medical strategy aiming these systems in the medical treatment of AR and expand the paradigm in the study of this form of LV volume overload.

Usefulness of Carvedilol in the Treatment of Chronic Aortic Valve Regurgitation

Background—Aortic regurgitation (AR) is a chronic disease for which there is currently no approved medical treatment. We previously reported in an animal model that &bgr;-blockade with metoprolol exerted beneficial effects on left ventricular remodeling and survival. Despite the recent publication of promising human data, &bgr;-blockade in chronic AR remains controversial. More data are needed to support this potentially new treatment strategy. We hypothesized that carvedilol might be another safe treatment option in chronic AR, considering its combined &bgr;-blocking and &agr;-blocking effects and proven efficacy in patients with established heart failure. Methods and Results—The effects of a 6-month treatment with carvedilol 30 mg/kg/d orally were evaluated in adult Wistar rats with severe AR. Sham-operated and untreated AR animals were used as controls. Carvedilol treatment resulted in less left ventricular hypertrophy and dilatation. Ejection fraction was improved and filling pressures were reduced by carvedilol. &bgr;1-Receptor expression was also improved as well as myocardial capillary density. Those beneficial effects were noted despite the presence of drug-induced bradycardia. Conclusions—Carvedilol exerted protective effects against volume-overload cardiomyopathy in this model of aortic valve regurgitation with preserved ejection fraction. These results suggest a protective class effect of &bgr;-blockers. Combined with the recent publication of promising human data, our findings support the need to carefully design a prospective study in humans to evaluate the effects of &bgr;-blockers in chronic aortic valve regurgitation.

Comparative Study of Vasodilators in an Animal Model of Chronic Volume Overload Caused by Severe Aortic Regurgitation

Background—Aortic regurgitation (AR) is a disease of chronic left ventricular (LV) volume overload. Over time, AR will lead to LV dilatation, hypertrophy, and loss of function. There is currently no medical treatment proven effective to slow the evolution of this cardiomyopathy. Vasodilators were once thought to have protective effects, but recent publications have cast some doubts about their effectiveness. We hypothesized that drugs targeting the renin-angiotensin system should be more effective than those having no direct effect on the renin-angiotensin system. Methods and Results—We designed a protocol comparing the effects of 3 vasodilators in a rat AR model (n=9 to 11 animals per group). The effects of a 6-month treatment of (1) nifedipine, (2) captopril, or (3) losartan were compared in male AR rats. Sham-operated and untreated AR animals were used as controls. Nifedipine-treated animals displayed hemodynamics, LV dilatation, hypertrophy, and loss of function similar to those of the untreated group. Both captopril and losartan were effective in improving hemodynamics, slow LV dilatation, hypertrophy, and dysfunction. Gene expression analysis confirmed the lack of effects of the nifedipine treatment at the molecular level. Conclusions—Using an animal model of severe AR, we found that vasodilators targeting the renin-angiotensin system were effective to slow the development of LV remodeling and to preserve LV function. As recently shown in the most recent human clinical trial, nifedipine was totally ineffective. Targeting the renin-angiotensin system seems a promising avenue in the treatment of this disease, and clinical trials should be carefully designed to re-evaluate the effectiveness of angiotensin I–converting enzyme inhibitors or angiotensin II receptor blockers in AR.

G2/M blockade by paclitaxel induces caveolin-1 expression in A549 lung cancer cells: caveolin-1 as a marker of cytotoxicity.

Caveolins are highly expressed in terminally differentiated cells, but this expression is down-regulated in various cancer cell lines. Exposure to low doses of paclitaxel (taxol) is sufficient to up-regulate caveolin-1, suggesting that a mild cytotoxic stress induces a response implying caveolin and caveolae. Here we show that this up-regulation is sustained even after the cessation of paclitaxel treatment. After exposure to a cytostatic dose of paclitaxel (50 nM), A549 lung cancer cells are blocked in the G2/M cell cycle phase. After removal of paclitaxel, cell death occurs, accompanied with an increase in caveolin expression, suggesting an effect of caveolin in this process. Three days post-paclitaxel treatment, surviving A549 cells were passaged and only a half of them adhered to the culture dish. Adhering cells (still mainly in the G2/M cell cycle phase) were still unable to grow and progressively entered in an apoptotic state. This study suggests that effects of a low dose of paclitaxel were still present even 1 week after drug removal and that caveolin-1 is a good marker of cytotoxicity.