Biomedical subjects

Philippe G Frank

Publications and source records attributed to Philippe G Frank.

10 recordsLinked to original sources

PubMed2003

Caveolin-1 null (-/-) mice show dramatic reductions in life span.

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.

Aging↗

PubMed2003

Genetic ablation of caveolin-1 confers protection against atherosclerosis.

OBJECTIVE: The development of atherosclerosis is a process characterized by the accumulation of lipids in the form of modified lipoproteins in the subendothelial space. This initiating step is followed by the subsequent recruitment and proliferation of other cell types, including monocytes/macrophages and smooth muscle cells. Here, we evaluate the potential role of caveolae membrane domains in the pathogenesis of atherosclerosis by using apolipoprotein E-deficient (ApoE-/-) mice as a model system. METHODS AND RESULTS: Caveolin-1 (Cav-1) is a principal structural protein component of caveolae membrane domains. To directly assess the in vivo role of caveolae and Cav-1 in atherosclerosis, we interbred Cav-1-/- mice with ApoE-/- mice. Interestingly, loss of Cav-1 resulted in a dramatic >2-fold increase in non-HDL plasma cholesterol levels in the ApoE-/- background. However, despite this hypercholesterolemia, we found that loss of Cav-1 gene expression was clearly protective against the development of aortic atheromas, with up to an approximately 70% reduction in atherosclerotic lesion area. Mechanistically, we demonstrated that loss of Cav-1 resulted in the dramatic downregulation of certain proatherogenic molecules, namely, CD36 and vascular cell adhesion molecule-1. CONCLUSIONS: Taken together, our results indicate that loss of Cav-1 can counteract the detrimental effects of atherogenic lipoproteins. Thus, Cav-1 is a novel target for drug development in the pharmacologic prevention of atheroma formation. Our current data also provide the first molecular genetic evidence to support the hypothesis that caveolar transcytosis of modified lipoproteins (from the blood to the sub-endothelial space) is a critical initiating step in atherosclerosis.

Animals↗

PubMed2003

Caveolin, caveolae, and endothelial cell function.

Caveolae are 50- to 100-nm cell-surface plasma membrane invaginations observed in terminally differentiated cells. They are particularly abundant in endothelial cells, where they are believed to play a major role in the regulation of endothelial vesicular trafficking and signal transduction. The use of caveolin-1-deficient mice has provided many new insights into the roles of caveolae and caveolin-1 in the regulation of endothelial cell function. These novel findings suggest an important role for caveolin-1 in the pathogenesis of cancer, atherosclerosis, and vascular disease.

Arteriosclerosis↗

PubMed2003

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Animals↗

PubMed2002

Stabilization of caveolin-1 by cellular cholesterol and scavenger receptor class B type I.

Caveolae are 50-100 nm plasma membrane invaginations, which function in cell signaling, in transcytosis, and in regulating cellular cholesterol homeostasis. These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins. Recent studies have indicated that caveolae may be involved in the regulation of cellular cholesterol efflux to high-density lipoproteins (HDL), as well as selective cholesteryl ester uptake mediated by scavenger receptor class B type I (SR-BI). In the present studies, we show that caveolin-1 expression in HEK-293T cells has no effect on SR-BI-mediated cellular cholesterol efflux to reconstituted HDL. However, SR-BI-mediated selective cholesteryl ester uptake is significantly inhibited by caveolin-1. Interestingly, we also found that SR-BI, but not CD36, can induce the dramatic stabilization of the caveolin-1 protein, independently of its transcriptional control. On the other hand, caveolin-1 has little effect on SR-BI stability, but clearly increases CD36 stability. Since SR-BI expression has been shown to increase cellular cholesterol levels, we next examined the effect of cholesterol itself on caveolin-1 stabilization and localization. When cells were loaded with cholesterol, we observed the dramatic stabilization of caveolin-1 with significant clustering of caveolin-1 at the cell surface. In addition, a palmitoylation-deficient caveolin-1 mutant was still responsive to cholesterol-induced stabilization, indicating that palmitoylation of caveolin-1 is not required for the cholesterol-induced stabilization of caveolin-1. These results suggest an important role for cholesterol and SR-BI in the regulation of caveolin functioning, especially in cell types such as endothelial cells and macrophages, which can be dramatically affected by changes in their cholesterol content during the development of atherosclerosis.

Animals↗

PubMed2002

Microvascular hyperpermeability in caveolin-1 (-/-) knock-out mice. Treatment with a specific nitric-oxide synthase inhibitor, L-NAME, restores normal microvascular permeability in Cav-1 null mice.

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (-/-) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis. Thus, a compensatory mechanism must be operating in these mice. One possible compensatory response would be an increase in the paracellular pathway, resulting in increased microvascular permeability. To test this hypothesis directly, we studied the microvascular permeability of Cav-1 null mice using a variety of complementary in vivo approaches. Radio-iodinated bovine serum albumin was injected into Cav-1-deficient mice, and its rate of clearance from the circulatory system was compared with that of wild type control mice. Our results indicate that iodinated bovine serum albumin is removed from the circulatory system of Cav-1-deficient mice at a substantially faster rate. To determine whether this defect is restricted to the paracellular movement of albumin, lungs from Cav-1-deficient mice were next perfused with the electron dense dye Ruthenium Red. Micrographs of lung endothelial cells from Cav-1-deficient mice demonstrate that the paracellular movement of Ruthenium Red is dramatically increased. In addition, electron micrographs of Cav-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane. This defect in cell-substrate attachment is consistent with the postulated role of caveolin-1 in positively regulating integrin signaling. Because loss of caveolin-1 expression results in constitutive activation of eNOS activity, we also examined whether these increases in microvascular permeability are NO-dependent. Interestingly, treatment with l-NAME (a well established nitric-oxide synthase inhibitor) successfully reversed the microvascular hyperpermeability phenotype of Cav-1 knock-out mice. Thus, caveolin-1 plays a dual regulatory role in controlling microvascular permeability: (i) as a structural protein that is required for caveolae formation and caveolar transcytosis and (ii) as a tonic inhibitor of eNOS activity to negatively regulate the paracellular pathway.

Animals↗

PubMed2002

Heteronuclear NMR studies of human serum apolipoprotein A-I. Part I. Secondary structure in lipid-mimetic solution.

The apolipoprotein A-I (apoA-I) solution structure in the presence of sodium dodecyl sulfate (SDS) was determined by combination of chemical shift index and torsion angle likelihood obtained from shift and sequence similarity methods. ApoA-I in lipid-mimetic solution is composed of alpha-helices (residues 8-32, 45-64, 67-77, 82-86, 90-97, 100-118, 122-140, 146-162, 167-205, 210-216 and 221-239), with 2-5 residue irregular segments between helical repeats, and the irregular segment 78-81 within helical repeat 2. ApoA-I is a monomer in the SDS complex and no evidence of interhelical interactions is found. Comparison of the apoA-I and apoA-I(1-186) [Okon et al., FEBS Lett. 487 (2001) 390-396] solution structures revealed that apoA-I undergoes a conformational change around Pro121.

Apolipoprotein A-I↗

PubMed2002

Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype.

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Adipocytes↗

PubMed2002

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade.

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

Amino Acid Sequence↗

PubMed2001

Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities.

Caveolae organelles and caveolin-1 protein expression are most abundant in adipocytes and endothelial cells. Our initial report on mice lacking caveolin-1 (Cav-1) demonstrated a loss of caveolae and perturbations in endothelial cell function. More recently, however, observation of the Cav-1-deficient cohorts into old age revealed significantly lower body weights, as compared with wild-type controls. These results suggest that Cav-1 null mice may have problems with lipid metabolism and/or adipocyte functioning. To test this hypothesis directly, we placed a cohort of wild-type and Cav-1 null mice on a high fat diet. Interestingly, despite being hyperphagic, Cav-1 null mice show overt resistance to diet-induced obesity. As predicted, adipocytes from Cav-1 null null mice lack caveolae membranes. Early on, a lack of caveolin-1 selectively affects only the female mammary gland fat pad and results in a near complete ablation of the hypo-dermal fat layer. There are also indications of generalized adipose tissue pathology. With increasing age, a systemic decompensation in lipid accumulation occurs resulting in dramatically smaller fat pads, histologically reduced adipocyte cell diameter, and a poorly differentiated/hypercellular white adipose parenchyma. To gain mechanistic insights into this phenotype, we show that, although serum insulin, glucose, and cholesterol levels are entirely normal, Cav-1 null mice have severely elevated triglyceride and free fatty acid levels, especially in the post-prandial state. However, this build-up of triglyceride-rich chylomicrons/very low density lipoproteins is not due to perturbed lipoprotein lipase activity, a major culprit of isolated hypertriglyceridemia. The lean body phenotype and metabolic defects observed in Cav-1 null mice are consistent with the previously proposed functions of caveolin-1 and caveolae in adipocytes. Our results show for the first time a clear role for caveolins in systemic lipid homeostasis in vivo and place caveolin-1/caveolae as major factors in hyperlipidemias and obesity.

Adipocytes↗