About Author:
Mr. Piyush Tripathi
Kota College of Pharmacy,
Kota (RAJ).
piyushtripathi1992@rediffmail.com
Introduction:
In biology, caveolae (Latin for little caves, singular: caveola), which are a special type of lipid raft, are small (50–100 nanometer) invaginations of the plasma membrane in many vertebrate cell types, especially in endothelial cells and adipocytes.
These flask-shaped structures are rich in proteins as well as lipids such as cholesteroland sphingolipids and have several functions in signal transduction. They are also believed to play a role in endocytosis, oncogenesis, and the uptake of pathogenic bacteria and certain viruses.
REFERENCE ID: PHARMATUTOR-ART-1847
Caveolins:
Formation and maintenance of caveolae is primarily due to the protein caveolin, a 21 kD protein. There are three homologous genes of caveolin expressed in mammalian cells: Cav1, Cav2 and Cav3. These proteins have a common topology: cytoplasmic N-terminus with scaffolding domain, long hairpin transmembrane domain and cytoplasmic C-terminus. Caveolins are synthesized as monomers and transported to Golgi apparatus. During their subsequent transport through secretory pathway, caveolins associate with lipid rafts and form oligomers (14-16 molecules). These oligomerized caveolins form the caveolae. The presence of caveolin leads to the local change in morphology of the membrane.
Roles of Caveolae:
- Caveolae can be used for entry to the cell by some pathogens and so they avoid degradation in lysosomes. However, some bacteria do not use typical caveolae but only caveolin-rich areas of the plasma membrane. The pathogens exploiting this endocytic pathway include viruses such as SV40 and polyoma virus and bacteria such as some strains of Escherichia coli, Pseudomonas aeruginosa and Porphyromonas gingivalis.
- Caveolae have a role in the cell signaling, too. Caveolins associate with some signaling molecules (e.g. eNOS) through their scaffolding domain and so they can regulate their signaling. Caveolae are also involved in regulation of channels and in calcium signaling.
- Caveolae also participate in lipid regulation. High levels of caveolin Cav1 are expressed in adipocytes. Caveolin associates with cholesterol, fatty acids and lipid droplets and is involved in its regulation.
- Caveolae can also serve as mechanosensors in various cell types. In endothelial cells, caveolae are involved in flow sensation. Chronic exposure to the flow stimulus leads to increased levels of caveolin Cav1 in plasma membrane, its phosphorylation, activation of eNOS signaling enzyme and to remodeling of blood vessels. In smooth-muscle cells, caveolin Cav1 has a role in stretch sensing which triggers cell-cycle progression.
Role of Caveolae and Caveolins in Health and Disease:-
Although they were discovered more than 50 years ago, caveolae have remained enigmatic plasmalemmal organelles. With their characteristic “flasklike” shape and virtually ubiquitous tissue distribution, these interesting structures have been implicated in a wide range of cellular functions. Similar to clathrin-coated pits, caveolae function as macromolecular vesicular transporters, while their unique lipid composition classifies them as plasma membrane lipid rafts, structures enriched in a variety of signaling molecules. The caveolin proteins (caveolin-1, -2, and -3) serve as the structural components of caveolae, while also functioning as scaffolding proteins, capable of recruiting numerous signaling molecules to caveolae, as well as regulating their activity. That so many signaling molecules and signaling cascades are regulated by an interaction with the caveolins provides a paradigm by which numerous disease processes may be affected by ablation or mutation of these proteins. Indeed, studies in caveolin-deficient mice have implicated these structures in a host of human diseases, including diabetes, cancer, pulmonary fibrosis, and a variety of degenerative muscular dystrophies.
The Multiple Faces Of Caveolae:-
Caveolae are a highly abundant but enigmatic feature of mammalian cells. They form remarkably stable membrane domains at the plasma membrane but can also function as carriers in the exocytic and endocytic pathways. The apparently diverse functions of caveolae, including mechanosensing and lipid regulation, might be linked to their ability to respond to plasma membrane changes, a property that is dependent on their specialized lipid composition and biophysical properties.
Caveolae-Associated Signaling In Health And Disease:-
Lipid rafts are planar aggregates of specific lipids that organize the membrane into domains with unique phase behaviours. Caveolae, considered a subcategory of lipid rafts, are 60-80 nm membrane invaginations stabilised by caveolin and PTRF-Cavin. Both microdomains are considered to harbour key signal transduction molecules. The overall aim of the present proposal is to unveil the function of caveolae and lipid rafts in the cardiovascular and urogenital systems. To do this, genetically modified animals, with perturbed function of receptors and proteins of relevance for formation and trafficking of caveolae/rafts, are utilized. An additional approach is depletion of membrane cholesterol, which disrupts both microdomains. Albeit less specific, this allows critical comparisons with human tissue.
Specific aims include-
i) to assess how receptor signaling is influenced by caveolae,
ii) to examine what role PTRF-Cavin plays in arterial function,
iii) to test whether caveolae-dependent trafficking affects vascular function, and
iv) to probe the possibility that bladder over activity following outlet obstruction is due to increased caveolae density.
The studies will provide in-depth understanding of the role of caveolae in cardiovascular and urogenital physiology. Novel therapeutic targets for treatment of hypertension, atherogenesis, erectile-, and bladder dysfunction may be discovered.
The Role Of Caveolae In Endothelial Cell Dysfunction With A Focus On Nutrition And Environmental Toxicants:-
Complications of vascular diseases, including atherosclerosis, are the number one cause of death in Western societies. Dysfunction of endothelial cells is a critical underlying cause of the pathology of atherosclerosis. Lipid rafts, and especially caveolae, are enriched in endothelial cells, and down-regulation of the caveolin-1 gene may provide protection against the development of atherosclerosis. There is substantial evidence that exposure to environmental pollution is linked to cardiovascular mortality, and that persistent organic pollutants can markedly contribute to endothelial cell dysfunction and an increase in vascular inflammation. Nutrition can modulate the toxicity of environmental pollutants, and evidence suggests that these affect health and disease outcome associated with chemical insults. Because caveolae can provide a regulatory platform for pro-inflammatory signaling associated with vascular diseases such as atherosclerosis, we suggest a link between atherogenic risk and functional changes of caveolae by environmental factors such as dietary lipids and organic pollutants. For example, we have evidence that endothelial caveolae play a role in uptake of persistent organic pollutants, an event associated with subsequent production of inflammatory mediators. Functional properties of caveolae can be modulated by nutrition, such as dietary lipids (e.g. fatty acids) and plant-derived polyphenols (e.g. flavonoids), which change activation of caveolae associated signaling proteins. The following review will focus on caveolae providing a platform for pro-inflammatory signaling, and the role of caveolae in endothelial cell functional changes associated with environmental mediators such as nutrients and toxicants, which are known to modulate the pathology of vascular diseases.
The Cell Surface in Health and Disease :-
The properties of the plasma membrane rely on the specialisation of the plasma membrane into microdomains of specific function. We are studying two types of surface microdomains: caveolae, a specialised domain of the cell surface with a distinct structure, and clathrin-independent endocytic carriers (CLICs), that form the major endocytic pathway in mammalian cells. Caveolae have been implicated in regulation of cell growth and in maintaining the balance of lipids in the cell. In addition, caveolins, the major membrane proteins of caveolae, and cavins, a newly discovered family of caveolar coat proteins, have been implicated in a number of disease states including tumour formation, lipodystrophies, and muscular dystrophy. To study caveolae function, we are using caveola-null mice, cells lacking caveolins and/or cavins, and zebrafish embryos. These systems are also being used to study the role of caveolae in muscle and the molecular changes associated with muscular dystrophy.
Lipid Rafts and Caveolae in the Terminal Differentiation of Epidermal Keratinocytes:-
The epidermal barrier resides in the protective, semipermeable stratum corneum (SC) that permits terrestrial life. An intact SC is crucial to maintain a barrier that prevents the loss of fluids, electrolytes and other molecules from within the body and, at the same time, prevents penetration by microorganisms, toxic materials and UV radiation. SC permeability barrier function is provided by lipid bilayer lamellae surrounding apoptotic corneocytes, the so-called “bricks-and-mortar model”. The intercellular spaces are filled with lipid lamellae build from a mixture of ceramides, free sterols, and free fatty acids made by the secretion of lamellar bodies (LB) at the level of the stratum corneum/stratum granulosum (SC/SG) junction. LB originates from the tubulo-vesicular elements of the trans- Golgi network, where lipids and proteins are sorted for secretion.
THE DYNAMICS OF LAMELLAR BODIES SECRETION-
LB secreted at the SC/SG junction, fuse with the apicalplasma membrane (APM) of the outermost SG cell, creatingthus a cholesterol/glycosphingolipid-enriched lipid raft-like domain. This secretion happens at low rates under normal conditions allowing a sufficient delivery of LB content, enough to maintain barrier function. However, immediately following acute barrier abrogation, an orchestrated sequence of responses occurs rapidly to restore the barrier function to its basal level. Among these, the instant secretion (within 30 minutes) of the preformed LB from the outermost SG takes place.
Application of either monensin or brefeldin A, known inhibitors of exocytosis and organellogenesis delay barrier recovery by affecting LB secretion and content respectively. While the signaling events that regulate LB formation/secretion are not yet fully understood, a decline in cation gradients across the epidermis (i.e. calcium and potassium) stimulates the initial secretion of LB that occurs in response to barrier disruption. Nevertheless, the secreted lipids “dumped” from LB-fusion with the AMP at the SG/SC are processed into lipid bilayers by secretory phopholipase A2 (sPLA2), steroidsulfatase (SSase), acid sphingomyelinase (aSMase) and glucocerebrosidase in the SC. Surprisingly, we recently found that aSMase delivered to the SC interstices is expressed in the raft domain fraction of the epidermis.
THE LIPID RAFT HYPOTHESIS-
Simons and van Meer back in 1988 described lipid rafts domains as dynamic, localized assemblies of cholesterol and sphingolipids within the plasma membrane. Caveolae represent a subclass of those rafts and are enriched in caveolin proteins, a family of three (cav-1 to -3) small molecular weight (18-24 kDa) proteins, that cycle between the trans-Golgi network and the plasma membrane (i.e. the natural flow of LB). Caveolin proteins form homo- and hetero-oligomers, which directly bind to cholesterol, required for the insertion of caveolae into membranes. Cav-1 possesses a ‘scaffolding domain’ that interacts with signal transduction molecules. Not only is this domain required to form multivalent homo-oligomers with other cav proteins, but it also mediates the interaction of cav-1 with non-cav proteins, such as the G-subunits, Ha-Ras, Src family kinases and eNOS. Consequently, cav-1 acts as molecular ‘Velcro’ where signal transduction complexes are bound in the inactivated state. Among the 3 cav proteins, cav-1 has been reported to be essential for caveolae formation. However, neither caveolin (1-3) knockout (-/-) mice show major abnormalities in their phenotype.
Many functions have been attributed to lipid rafts/ caveolae, and recently a role of caveolin-1 in lamellar body assembly, trafficking and function was suggested. This could indicate a role of cavolin-1 and caveolae formation in epidermal barrier permeability
homeostasis. Studies in methyl-cyclodextrin-treated mice show that disruption of lipid rafts leads to alterations in plasma membrane dynamics necessary for adequate lamellar body secretion at the SG/SC interface. In addition, studies in caveolin-1 knockout mice and monensin-treated mice demonstrate the importance of caveolin-1insertion into lipid rafts and caveolae formation in barrier restoration. Next to an
important role of caveolae in lamellar body secretion and terminal differentiation, preliminary data also suggest a role in establishing cell-cell contacts and adherens junction formation.
Age-Related Changes of Caveolin-1 Expression : A New Role for Caveolins:-
In biological terms, caveola are a specialized type of small invaginations (50-100 nanometers) in the plasma membrane of several vertebrate cells that participate in the regulation of a considerable quantity of cellular functions. At cardiovascular level, they are present in almost all cardiac cells including smooth muscle cells, endothelial cells, myocyte, fibroblasts and macrophages. Caveola were discovered between 1953 and 1955 by Palade and Yamada, who demonstrated the presence of gallbladders that were not related to clathrin in endothelial cells and epithelial cells of the gallbladder. Since then, these cellular structures were studied in order to know their normal functioning and, more recently, in which way their alterations are involved in different pathologies. The organization and function of caveola are given by coat proteins, called caveolins, and adaptation proteins, called cavins. Caveolin, with its three isoforms (caveolin-1, caveolin-2, and caveolin-3), form the backbone and are highly integrated in their function. Caveolin-1 and 2 are present in most of the cardiovascular system cells, while caveolin-3 is present in the smooth muscle, striated and cardiac cells. On the other hand, cavins act as regulators of caveolin functioning.
Functionally, caveola participate in cellular signalling and in the regulation of vesicular transport kinetics, fulfilling in this way numerous activities. Signalling function is produced thanks to the high concentration of receptors and intracellular molecules in the place of invagination, which allow an efficient signal transduction. Among other functions, caveola are inhibitors of the activity of the endothelial enzyme nitric oxide synthase (eNOS) while interacting and form a complex eNOS/caveolin-1 that decreases the formation of nitric oxide (NO). In this way, caveolin-1 is an important regulator of NOS functioning. Alterations of these proteins, in different pathologies, produce modifications in NO metabolism, as it is the case of diabetes, in which the overexpression of caveolin-1 generates a negative regulation of the eNOS activity. In the same way it was demonstrated that aging is associated with an increase in the expression of caveolin-1 in human fibroblasts and the reduction of these caveolins in senescent fibroblasts is able to reverse its phenotype to a level of activity similar to the one of young cells.
In situations of hypovolemia, the activation of NO system during the haemorrhage is an important compensatory mechanism. In this sense, in this current issue of the Argentine Journal of Cardiology, considered an interesting hypothesis while studying in which way this system of adaptation could be altered with the advancing age and they try to explain these changes with the modifications in caveolin functioning. The authors demonstrate that there is a lower expression of eNOS in the group of adult animals compared with young ones. This was not correlated with the enzyme activity, as it was expressed by the authors, eNOS activity was similar both in adult and young animals. This last piece of information is opposed to the idea of aging associated with an eNOS negative regulation while increasing the expression of caveolin-1. This controversy makes Arreche et al. results more interesting, while arranging the caveolin association/dissociation phenomenon with the eNOS, as although having lower expression of protein, the activity is not modified, probably as a dissociation of the eNOS with caveolin. However, in their study, the authors did not measure the expression and the activity of caveolin-1, which would have helped to answer, at least partially, this question. Surely, this question would be answered in future works.
On the other hand, a state of hypovolemia affects not only to myocyte, but also the vascular component. In this sense, caveola are important regulators of the vascular tone, due to their capacity of modulating eNOS activity. Therefore, it would be interesting to evaluate the eNOS answer, at vascular endothelial level, during acute haemorrhage and also considering aging.
It is clear that there are many questions concerning this interesting topic, particularly in which way could some pathologic states that are often associated with aging as diabetes or hypertension in the adaptive response of NO, associated with hypovolemia and in relation with caveola activity affect. This is important as there is a growing interest about the role of caveola and its structural protein, caveolin-1, in the normal and pathological functioning of the cardiovascular system.
The Role of Caveolin-1 in Prostate Cancer: Clinical Implications:-
Caveolin-1 (cav-1) is a major structural component of caveolae, which are specialized plasma membrane invaginations involved in multiple cellular processes such as molecular transport, cell adhesion and signal transduction. Although under some conditions cav-1 may suppress tumorigenesis, cav-1 is associated with and contributes to malignant progression through various mechanisms. Specific proteins such as receptor tyrosine kinases, serine/threonine kinases, phospholipases, G protein-coupled receptors and Src family kinases are localized in lipid rafts and caveolar membranes, where they interact with cav-1 through the cav-1 scaffolding domain; the activities mediated by this domain result in the generation of platforms for compartmentalization of discrete signaling events. A high level of intracellular cav-1 expression is associated with metastatic progression of human prostate cancer and other malignancies, including lung,renaland esophageal squamous cell cancers.
Virulent prostate cancer cell lines reportedly secrete biologically active cav-1 protein in vitro, and cav-1 promotes prostate cancer cell viability and clonal growth. The cancer-promoting effects of secreted cav-1 include antiapoptotic activities similar to those observed following enforced expression of cav-1 within the cells. In addition to showing cav-1-mediated autocrine activities, a recent study showed that recombinant cav-1 protein is taken up by prostate cancer cells and endothelial cells in vitro and that recombinant cav-1 increases angiogenic activities both in vitro and in vivo by activating Akt- and/or nitric oxide synthase-mediated signaling. Moreover, significantly higher serum cav-1 levels have been documented in men with prostate cancer than in men with benign prostatic hyperplasia and also in patients with elevated risk of cancer recurrence after radical prostatectomy.
The concept of expression and secretion of cav-1 by prostate cancer cells in malignant progression is unique. The autocrine and paracrine activities of cav-1 mediated through the activation of Akt and/or nitric oxide synthase signaling may lead to pervasive engagement of the local tumor microenvironment, involving but not limited to the proangiogenic activities previously documented.
Mechanisms of Cav-1-mediated Oncogenic Activities in Prostate Cancer:-
Overexpression of cav-1 was reported in various malignancies, including cancer of the colon, kidney,bladder, lung, pancreas and ovary, and in some types of breast cancer. The level of cav-1 expression may depend on the tumor type and stage; for example, high cav-1 levels were reported in late or advanced squamous cell carcinoma and in metastatic prostate cancer. These results have led many investigators to attempt to identify cav-1-related oncogenic pathways for various malignancies. Although cav-1 activities impinge on various oncogenic pathways and can inhibit or activate these pathways, depending on the cell type and context, the results of multiple studies now indicate that Akt activation has an important role in cav-1-mediated oncogenic functions in prostate cancer. The first demonstration of a direct association between cav-1 expression and Akt indicated that the overexpression of cav-1 increased binding to and inhibited the serine/threonine phosphatases, PP1 and PP2A, in human prostate cancer cells. These interactions, which were likely mediated through cav-1 binding to a cav-1 scaffolding domain-binding site on PP1 and PP2A and inhibition of their activities, led to significantly increased levels of phospho-Akt and sustained activation of downstream oncogenic Akt targets. Findings from a recent independent study supported this mechanism and further showed that the putative oncogene inhibitor of differentiation-1 induced Akt activation by promoting the binding activity of cav-1 and PP-2A. It is important to consider that the activation of Akt has been previously associated with prostate cancer and is clearly one of the most important oncogenic activities that underlie prostate cancer progression. It is worthwhile to consider the idea that activated Akt contributes to the expression and secretion of multiple growth factors (GFs) that have important roles in the growth, survival and progression of prostate cancer cells through autocrine and paracrine activities. The molecular mechanisms that may connect cav-1 upregulation to GF expression and secretion through the activation of Akt are worthy of future investigation.
Secreted Cav-1 as a Biomarker and Therapeutic Target:-
The expression and secretion of cav-1 by prostate cancer cells presents an opportunity for the development of cav-1-based biomarkers for prostate cancer. We previously developed an immunoassay for measuring serum cav-1 levels and showed that the median serum cav-1 level in men with clinically localized prostate cancer was significantly higher than that in healthy control men (that is, in those with normal findings on digital rectal examination and serum prostate-specific antigen levels of ≤1.5 ng ml−1 over a period of 2 years) and in men with clinical benign prostatic hyperplasia. Further, in a larger population study in men with a serum prostate-specific antigen of >10 ng ml−1, high pretreatment levels of cav-1 in the serum were associated with a shorter time to biochemical recurrence (defined as a serum prostate-specific antigen level of ≥0.2 ng ml−1 on two consecutive measurements). High pretreatment serum cav-1 levels were established using a cutoff determined by using the minimum P-value method.
These initial clinical and basic laboratory study results, together with those of pathology-based tissue analysis, show the potential of serum cav-1 as a prognostic biomarker for the identification of men with clinically aggressive prostate cancer. Specifically, the pretreatment serum cav-1 concentration may be used to identify men with clinically significant prostate cancer who are likely to experience a rapid recurrence of the cancer following radical prostatectomy. Although further studies are necessary to validate these results, it is conceivable that serum cav-1 analysis would contribute to the identification of the subset of the men undergoing localized therapy for presumed localized disease who would benefit from neoadjuvant or adjuvant therapy, for example, local radiotherapy, localized biologic therapy, androgen-deprivation therapy and/or targeted systemic therapy.
We have considered, in addition to the potential use of serum cav-1 analysis as a prognostic biomarker for clinically aggressive prostate cancer, the possibility that secreted cav-1 is a therapeutic target for prostate cancer. Our recent studies revealed that systemic treatment of mice with cav-1 antisera significantly reduced the development and growth of primary site tumors and metastases in both orthotopic and experimental metastasis mouse models of prostate cancer. These studies further showed that metastatic prostate cancer cells may survive and grow partly through the uptake of secreted cav-1. As targeted systemic antibody therapy has been used successfully to treat specific malignancies, the development of cav-1-targeted antibody therapy should be further pursued as a potential therapy for prostate cancer.
Cell-Specific Dual Role of Caveolin-1 in Pulmonary Hypertension:
Pulmonary hypertension (PH) is a rare but a devastating disease with high morbidity and mortality rate. The reported prevalence is 15–52 cases/million and the incidence is thought to be 2.4–7.6 cases/million/year. A wide variety of cardiopulmonary diseases, collagen vascular and autoimmune diseases, chronic thromboembolism, HIV, portal hypertension, drug toxicity, and myeloproliferative diseases are known to lead to PH. In primary pulmonary arterial hypertension (PAH), currently labeled as idiopathic PAH, the underlying etiology is not clear and about 6% of patients in this group have a family history of the disorder. Multiple signaling pathways and inflammation have been implicated in the pathogenesis of PH. Endothelial dysfunction may be an important triggering factor leading to an imbalance between vasorelaxation and vasoconstriction and deregulation of cell proliferation leading to vascular remodeling and PH with subsequent cell migration and neointima formation. Loss of bioavailability of nitric oxide (NO) and prostacyclin (PGI2) , upregulation/activation of proliferative molecules such as endothelin-1 (ET1) , platelet-derived growth factor (PDGF) , serotonin, survivin, cyclin D1, tyrosine-phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) , RhoA/Rho kinase, and anti-apoptotic molecules such as Bcl2 and Bcl-xLhave been reported in PH. In addition, increased elastase activityand increased production of matrix metalloproteinase2 (MMP2) occur in PH. Recent studies have shown a strong link between heterozygous germline mutations in bone morphogenic protein receptor type II (BMPRII), a member of TGFβ superfamily and pulmonary arterial hypertension (PAH). Mutation of BMPRII has been reported in 70% of heritable PAH, 26% IPAH, and 6% of patients with congenital heart defect and associated PAH. However, only about 20% of people with this mutation develop PAH, indicating that environmental and/or other genetic factors may be involved in the development of the disease. Furthermore, recent studies have shown reduction in the expression of BMPRII protein in both monocrotaline (MCT) and the hypoxia models of PH. In addition, mutations of activin-like receptor kinase 1 (ALK1) and endoglin, both belonging to TGFβ superfamily, have been reported in patients with hereditary hemorrhagic telangiectasia, and some of these patients develop PAH.
Regardless of the underlying etiology, the main features are endothelial dysfunction, impaired vascular relaxation response, deregulated cell proliferation and impaired apoptosis, vascular remodeling, narrowing of the lumen, elevated PA pressure, and right ventricular hypertrophy with subsequent right heart failure and premature death. Despite major advances in the understanding of the disease process, a cure is not yet in sight. Current therapy has improved the quality of life but has not had a significant effect on the mortality rate. Loss of endothelial caveolin-1, a cell membrane protein is well documented in experimental and clinical forms of PH. Recent studies indicate that in addition to the loss of endothelial caveolin-1, there is enhanced expression of caveolin-1 in smooth muscle cells with proliferative activity and subsequent neointima formation. Thus, caveolin-1 may play a key role in the pathogenesis of PH, and its activity may depend on cell type and the disease stage.
Endothelial Caveolin-1 in Pulmonary Hypertension :-
Disruption of Endothelial Cell Membrane and Loss of Caveolin-1-
Loss of endothelial caveolin-1 has been reported in clinical and experimental forms such as monocrotaline (MCT) and myocardial infarction models of PH.The MCT model has been extensively studied to understand the pathogenesis of PH. A single subcutaneous injection of MCT in rats injures endothelial cells within 24–48 hrsand PH is observed at 10–14 days after MCT. In this model the disruption of endothelial caveolae associated with progressive loss of caveolin-1 occurring as early as 48 hrs after MCT, is a major feature seen before the onset of PH. In addition to the loss of caveolin-1, there is reduction in the expression of other endothelial cell membrane proteins known to colocalize with caveolin-1 such as Tie2 (endothelium-specific tyrosine kinase receptor of angiopoietin 1), platelet endothelial cell adhesion molecule (PECAM) 1, and both subunits of soluble guanylate cyclase. Importantly, the loss of caveolin-1 is associated with reciprocal activation of signal transducer and activator of transcription (STAT) 3 to PY-STAT3, known to be preferentially activated by downstream effectors of proinflammatory cytokine IL-6/gp130 signaling pathway. In addition, the expression of Bcl-xL is increased simultaneously with the activation of PY-STAT3. PY-STAT3 plays a critical role in cell growth, inhibition of apoptosis, survival, and in immune function and inflammation. Persistent phosphorylation of STAT3 has been reported in a number of primary tumors, and activation of STAT3 signaling confers resistance to apoptosis.Some of the downstream effectors of PY-STAT3 are survivin and Bcl-xL (antiapoptotic factors), and cyclin D1 (cell-cycle regulator). All these factors have been shown to be upregulated in PH. Importantly, activation of PY-STAT3 has been observed in endothelial cells obtained from patients with idiopathic PAH. RhoA/Rho kinase activation is well established in PH, and interestingly, Rho GTPases is required for STAT3 activation, and Rho GTPases-mediated cell proliferation and migration occur via STAT3. Caveolin-1 functions as a suppressor of cytokine signaling (SOCS) 3 and inhibits PY-STAT3 activation. Therefore, it is not surprising that the rescue of endothelial caveolin-1 not only inhibits STAT3 activation but also restores the endothelial cell membrane integrity and attenuates MCT-induced PH and vascular remodeling. These results underscore the importance of endothelial cell membrane integrity and the expression of endothelial caveolin-1 in maintaining vascular health.
Studies with caveolin-2 KO mice have shown pulmonary defects such as alveolar wall thickening and increased cell proliferation similar to what has been reported in caveolin-1 KO mice. Unlike caveolin-1 KO, caveolin-2 KO has no effect on vascular reactivity, nor does it participate in the formation of caveolae. Interestingly, in the MCT and myocardial infarction models of PH, in addition to loss of caveolin-1, caveolin-2 loss occurs, and the rescue of caveolin-1 attenuates PH and also restores caveolin-2 expression. Since caveolin-2 requires caveolin-1 for its transport to the membrane surface, caveolin-2 loss may accompany the caveolin-1 loss in these models of PH. It is likely that caveolin-2 participates with caveolin-1 in pulmonary vascular health and disease. It is not clear what independent role caveolin-2 might have in the pathogenesis of PH. Further studies are warranted to examine the specific role of caveolin-2 in PH.
Perturbation of Endothelial Cell Membrane and Dysfunction of Caveolin-1-
PH is an important cause of heart failure and increased mortality in patients suffering from chronic lung diseases associated with alveolar hypoxia. Hypoxia induces pulmonary vasoconstriction and vascular remodeling leading to PH. In hypoxia-induced PH, similar to the MCT model, low bioavailability of NO, low basal and agonist-induced cGMP levels, and impaired endothelium-dependent NO-mediated relaxation responses in pulmonary arteries have been reported. Interestingly, BH4 or L-arginine administration does not improve eNOS dysfunction. However, unlike the MCT model, in hypoxia-induced PH, there is no reduction in caveolin-1 expression. Murata et al. have further shown that in pulmonary arteries from rats with hypoxia-induced PH, eNOS forms a tight complex with caveolin-1 and becomes dissociated from HSP90 and calmodulin, resulting in eNOS dysfunction. In addition, the long-term effect of prenatal hypoxia results in impaired endothelium-dependent and NO-mediated relaxation responses coupled with increased caveolin-1 and eNOS association. Interestingly, hypoxia-induced PH and pulmonary endothelial cells exposed to hypoxia exhibit hyperactivation of PY-STAT3. Hypoxia-inducible factor (HIF) 1α is thought to play a significant role in hypoxia-induced hyperplasia of SMC. STAT3 plays a significant role in stabilizing HIF1α, and its interaction with HIF1α mediates transcriptional activation of vascular endothelial growth factor (VEGF) promoter. Targeting STAT3 blocks HIF1α and VEGF, thus modulating proliferation and angiogenesis. These results strongly suggest that PY-STAT3 may be an important regulator of VSMC proliferation in PH irrespective of the underlying etiology.
Since caveolin-1 has been shown to inhibit PY-STAT3 activation, the activation of PY-STAT3 in hypoxia-induced PH despite the unaltered expression of caveolin-1 protein strongly suggests that caveolin-1 is dysfunctional and has lost its inhibitory function. Furthermore, within 24 hr exposure to hypoxia, bovine pulmonary artery endothelial cells reveal caveolin-1 and eNOS complex formation accompanied by PY-STAT3 activation. These results indicate that the tight complex formation of caveolin-1 and eNOS in hypoxia-induced PH renders both eNOS and caveolin-1 dysfunctional. In this context, it is worth noting that statins protect eNOS function in hypoxia-induced PH. The major effect of statins is reported to be the uncoupling of eNOS/caveolin-1 complex, thus freeing eNOS for activation. This effect on eNOS is not accompanied with lowering of cholesterol. It is likely that the statins disrupt the tight cavolin-1/eNOS coupling resulting from hypoxia- induced perturbation of endothelial cell membrane, thus restoring antiproliferative properties of caveolin-1 and NO production by eNOS. Unlike the MCT model, hypoxia does not appear to cause physical disruption of EC membrane but causes perturbation of the endothelial cell membrane and leading to “mislocalization” of caveolin-1 and eNOS.
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Dual Role of Caveolin-1-
Loss of caveolin-1 has been shown to induce oncogenic transformation, and the cells become resistant to apoptosis. Furthermore, the introduction of caveolin-1 scaffolding domain inhibits cancer progression. Many oncogenes transcriptionally downregulate caveolin-1 expression. However, caveolin-1 regulation impacts both negatively and positively on several aspects of tumor progression. Caveolin-1 acts as a tumor suppressor in the early stages of cancer, but in late stages it promotes metastasis, multidrug resistance, and portends poor prognosis. Caveolin-1 function is thought to be interdependent on tumor stage and the expression of molecular effectors that may have an impact on its role during tumor progression. Similarly in PH, the switch from an antiproliferative to proproliferative function may depend on alteration in caveolin-1 conformation, localization, cell context, and the stage of the disease.
Caveolae and caveolin-1 play an important role in pulmonary vascular system. Depending on the type of endothelial injury, the end result is either the loss of caveolin-1 secondary to endothelial cell membrane disruption or in endothelial caveolin-1 dysfunction. A classic example of the latter case is hypoxia-induced PH in which a tight complex formation of caveolin-1/eNOS resulting in dysfunction of both molecules is an important feature. Both these alterations, however, do lead to pulmonary vascular remodeling and PH. Disruption of endothelial cell membrane integrity as in the former case is often progressive leading to extensive EC damage and/or loss with subsequent enhanced expression of caveolin-1 in SMC, which participates in further proliferation, cell migration, and neointima formation. These alterations in caveolin-1 may determine reversibility versus irreversibility of the disease process. Thus, depending on the underlying pathology, cellular involvement, and the stage of the disease, modulation of caveolin-1 function may be considered a therapeutic target in PH.
Increased Smooth Muscle Cell Expression Of Caveolin-1 And Caveolae Contribute To The Pathophysiology Of Idiopathic Pulmonary Arterial Hypertension:-
Recent efforts to understand the pathogenesis and to develop treatments for IPAH have emphasized increased proliferation of PASMC leading to vascular wall hypertrophy and increased vascular tone as possible therapeutic targets. This “hyperproliferative” state has been linked to increased [Ca2+] levels in PASMC , but little is known regarding the precise molecular and cellular determinants. The data shown here involving assessment of mRNA, protein, ultrastructure, function, and molecular manipulation are all consistent with the idea that increased [Ca2+] in PASMC from IPAH patients is linked to overexpression of Cav-1 and caveolae. We found overexpression of Cav-1 in tissue sections and in PASMC cultured from patients with IPAH. In addition, two different interventions (MβCD and lovastatin) that deplete cholesterol and perturb the structure of caveolae decreased CCE and proliferation of IPAH-PASMC. We observed similar effects in IPAH-PASMC treated with siRNA directed against Cav-1. By contrast, overexpression of Cav-1 in normal PASMC increased formation of caveolae (to levels comparable to those found in IPAH) and recapitulated the enhanced CCE observed in IPAH-PASMC. Taken together, these multiple complementary pieces of evidence strongly implicate the expression of Cav-1 and caveolae in the regulation of [Ca2+] in IPAH-PASMC. The results suggest that statins (or perhaps other cholesterol-lowering agents) or therapies that reduce Cav-1 expression in PASMC may have therapeutic benefit for IPAH. However, such caveolae-targeted therapeutics must take into account the possibility of effects on other cell types; further data are needed to develop such a therapeutic approach.
Certain neoplasms [i.e., prostate cancer, bladder cancer, adenocarcinomas, esophageal squamous cell carcinomas, and both benign and malignant smooth muscle tumors] have elevated Cav-1 expression. Timme et al. have shown an interplay between Cav-1 and c-Myc-induced apoptosis: the Cav-1 gene can be down-regulated by c-myc, and maintaining high levels of Cav-1 suppresses c-myc-induced apoptosis. Cav-1 has also been shown to maintain active Akt in prostate cancer cells by inhibiting protein phosphatase activity, an additional mechanism that may contribute to cellular resistance to apoptosis. Such findings imply that, in IPAH, Cav-1 overexpression may be antiapoptotic and/or block proapoptotic pathways. Mutations in Cav-3 have been linked to a pathological process (i.e., muscular dystrophy). The current results indicate that another caveolin, Cav-1, contributes to pathology and may serve as both a marker and therapeutic target in IPAH.
Cav-1-deficient mice, which lack Cav-1 and -2 expression and caveolae formation, develop pulmonary hypertension, right ventricular hypertrophy, as well as structural remodeling, vasculopathies, and hyperproliferation in the lung. Recent data suggest that administration to rats of a cell-permeable Cav-1 peptide prevents monocrotaline-induced pulmonary hypertension. Such findings contrast with our findings in human IPAH, implying that the latter disease is not easily approximated in animal models. It is likely that the Cav-1-knockout mice develop pulmonary hypertension secondary to other pathologies. Expression of Cav-1 and Cav-2 is decreased in the lungs of rats with SPH, including monocrotaline- and myocardial infarction-induced pulmonary hypertension. Such data contrast with our results shown here for IPAH-PASMC, further demonstrating the difficulty of modeling the human disorder in studies with experimental animals. Limited data for patients with IPAH have utilized whole lung tissue and shown a reduction in caveolin mRNA and protein. When we analyzed tissue from whole lung, we, too, found a decrease in Cav-1 expression; however, assessment of specific cell types revealed a dramatic elevation of Cav-1 expression in vascular smooth muscle of IPAH patients with decreased expression in endothelial cells, the latter of which are likely the predominant contributors to analyses of caveolin expression in whole lung preparations. Our other data show that increased caveolin expression contributes to the altered smooth muscle cell function in patients with IPAH. One must therefore be cautious in extrapolating animal datato a complex human disease such as IPAH, which may show cell-specific changes such as those we observe in the expression of caveolins.
In the current study we show that increased caveolar formation increases CCE and [Ca2+]. [Ca2+] is tightly regulated: a rise in [Ca2+] is a trigger for vasoconstriction, and a stimulant of cell proliferation. [Ca2+] also regulates numerous enzymes. Depletion of Ca2+ from the SR leads to the opening of plasma membrane TRPC channels, thereby increasing Ca2+ influx (CCE), to refill the SR stores and allowing for sustained increase in cytoplasmic [Ca2+]. PASMC isolated from IPAH patients have higher [Ca2+] levels than do normal cells, likely attributable to up-regulation of TRPC that comprise the SOC. Increased expression of Cav-1 and caveolae in IPAH may contribute to this increased [Ca2+]viaup-regulation of TRPC expression and localization in caveolae. Disruption of caveolae via MβCD and lovastatin, as used in our study, may displace components of the Ca2+ signaling machinery from close association with SR whose proximity is necessary for activation of SOC in a local microenvironment. Since Cav-1 also localizes with BMPR2 and nitric oxide synthase, such interactions may also influence IPAH. The current findings provide complementary evidence from upward and downward manipulation of expression of Cav-1 to establish a molecular proof-of-principle for the role of enhanced Cav-1 expression and caveolae formation in altered Ca2+ handling and cellular responses in IPAH.
The therapeutic role of statins in PAH has been assessed in animals with hypoxia- and monocrotaline-induced pulmonary hypertension, but such studies do not reveal the cellular and molecular mechanisms for beneficial responses. Although statins have pleiotropic effects, the findings we obtained with PASMC imply that statins may act via cholesterol depletion and resultant decreased expression of caveolae; this mechanism will require further studies in animals that are administered such drugs. Our data showing that statins modulate CCE in IPAH-PASMC in parallel with altered membrane morphology and disruption of caveolae suggest that statins modify IPAH-PASMC physiology toward a nonproliferative phenotype linked to a decrease in [Ca2+].
Our results should be interpreted with certain limitations. Although the current data emphasize the increase in expression of Cav-1 in IPAH-PASMC, other data (not shown) indicate that expression of Cav-2 mRNA and protein are increased in IPAH-PASMC and that siRNA for Cav-1 also results in a down-regulation of Cav-2. The latter finding is consistent with reports in the literature indicating that Cav-1 is the dominant caveolin isoform that regulates cell physiology and that expression and localization of Cav-2 depend on the expression and localization of Cav-1. Such data suggest that Cav-1 is the more important isoform and are consistent with findings we obtained in siRNA and overexpression studies. In addition, we observed no expression of Cav-3 in either normal or IPAH-PASMC.
It is theoretically possible that the treatments administered to the IPAH patients we studied may have altered the expression of Cav-1 and caveolae; samples from untreated patients were not available to us. As noted above, it is possible that the beneficial effects we observed with statins are mediated independent of cholesterol reduction and, in turn, are independent of caveolae expression. Nevertheless, the current findings provide evidence for a previously unappreciated role of increased expression of Cav-1 and caveolae in the pathophysiology of IPAH and suggest that modifying their expression may have therapeutic benefit.
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