Caveolae In Endothelial Cells Research Articles

Effect of androgen replacement therapy on erectile function through histological recovery in castrated rats

ABSTRACT Introduction Androgens are essential for physical activity in males and normal erectile function. Reportedly, androgen deficiency causes erectile dysfunction through tissue damage in the corpus cavernosum. We previously reported that delayed androgen replacement therapy improved erectile function in rats through histological recovery. Objective To investigate the mechanism of the recovery process by androgen replacement therapy in rats. Methods Twelve-week-old male Wistar ST rats were used in this study. The rats were assigned to the following groups: castrated (Cast), castrated with subcutaneous administration of testosterone undecanoate (25 mg/kg; Cast+T), and sham (Sham). Cast+T rats received subcutaneous doses of testosterone, beginning from 4 weeks after castration, whereas Sham and Cast rats received only the vehicle. Erectile function was assessed by evaluating intracavernosal pressure (ICP) and mean arterial pressure (MAP) after electrical stimulation of the cavernous nerve at 1, 2, 4, and 8 weeks after testosterone administration. mRNA expression of histological factors (Cavin-1, Cavin-2, Cavin-3, α-SMA, VEGF-R, ICAM-1, and TGF-β1) was tested using real-time PCR. Results The ratio of ICP to MAP was 0.26 ± 0.04 in the 4th week for the Cast group, and the erectile function was significantly lower than that of the Sham group (0.61 ± 0.03; p <0.05). On the other hand, in the Cast+T group, it changed over time from 0.41 ± 0.06 in the 1st week, 0.44 ± 0.04 in the 2nd week, 0.48 ± 0.04 in the 4th week, to 0.70 ± 0.03 in the 8th week, and erectile function was significantly improved 8 weeks after the testosterone administration (p <0.05). The Cavin-1 and Cavin-2 mRNA expressions were increased in the Cast+T group compared to the Cast group at all times (P <0.01). α-SMA, VEGF-R, ICAM-1, and TGF-β1 mRNA expressions were also increased in the Cast+T group, as compared to the Cast group (P <0.01). Conclusions In this study, we identified that androgen replacement therapy in rats would improve ED. This may be because the administration of testosterone improved histological recovery. It has been reported that these caveolae-related factors control the invagination structure of the cell membrane, and that caveolae in endothelial cells are also involved in the production of nitric oxide (NO), which might contribute to the therapeutic effect of androgen replacement therapy. Increase in the expression of these caveolae-related factors may regenerate smooth muscle and corpus cavernosum structures. Disclosure Work supported by industry: no.

The Role of Major Facilitator Superfamily Domain-Containing 2a in the Central Nervous System.

Major facilitator superfamily-domain containing 2a (Mfsd2a) is selectively expressed in vascular endotheliocytes and plays a crucial role in maintaining the integrity of the blood‒brain barrier and the transport of docosahexaenoic acid. It is currently recognized as the only molecule that inhibits endocytosis mediated by caveolae in brain endothelial cells. Mfsd2a gene knockout leads to an increase in the permeability of the blood-brain barrier from embryonic stages to adulthood while maintaining the normal pattern of the vascular network. In Mfsd2a knockout mice, the docosahexaenoic acid content is significantly reduced and associated with neuron loss, resulting in microcephaly and cognitive impairment. Based on the role of Mfsd2a in the central nervous system, it has been preliminarily suggested as a potential therapeutic target for drug delivery to the central nervous system. This paper reviews the current progress in Mfsd2a research and summarizes the physiological functions of Mfsd2a in the central nervous system and its role in the occurrence and development of a variety of neurological diseases.

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.

Cavin-1 regulates BMP/Smad signaling through the interaction of Caveolin-1 with BMPRII in pulmonary artery endothelial cells

Abstract Background Pulmonary hypertension (PH) is a progressive disease associated with poor outcome. Caveolin-1 (Cav1) is a component of caveolae and classified as a related gene of pulmonary arterial hypertension (PAH). Gene mutations of bone morphogenetic protein type II receptor (BMPRII) is a most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Purpose The aim of our study is to investigate the relationship between Caveolin-Cavin system and BMP/Smad signaling pathway and explore the mechanism of downstream signal transduction of BMP signaling by the interaction between Caveolin and BMPRII. Methods Cav1 knockout mice were used to assess PH and caveolae in pulmonary artery endothelial cells were observed by electron microscope. Cav1 and Cavin-1, which is a component of caveolae and form a complex with Cav1, were knocked-down in human pulmonary artery endothelial cell (hPAEC) using siRNA and phosphorylation of Smad signal was evaluated. Apoptosis of these cells was explored by flow cytometry. We investigated the interaction between Cav1 and BMPRII, and evaluated whether Cavin-1 affects this interaction and signal transduction of BMP signaling. Results As previously described, deletion of Cav1 revealed disappearance of caveolae in pulmonary artery endothelial cells (PAECs), and Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy. We then examined roles of Cav1 in human PAECs (hPAECs). Cav1 knockdown in hPAECs reduced phosphorylation of Smad 1/5/9. In addition, Cav1 knockdown significantly increased hypoxia-induced apoptosis in hPAEC. Knockdown of Cavin-1 reversed phosphorylation of Smad 1/5/9 decreased by Cav1 knockdown in BMP9 stimulation. Cavin-1 reversed the expression of BMPRII decreased by overexpression of Cav1. Cav1 was associated with Cavin-1 at the plasma membrane in PAECs. Cav1 also associated with BMPRII at the membrane of hPAECs that was inhibited by Cavin-1, and Cavin-1 reduced the localization of BMPRII to the membrane of hPAECs. These results suggest that BMPRII interacts with Cav1 via Cavin-1-associated localization at the plasma membrane in hPAECs, resulting in regulating BMP/Smad signaling pathway and involving in the development of PAH. Conclusions Cavin-1 affects the interaction of Cav1 with BMPRII at the membrane of PAECs, and regulates BMP/Smad signaling. These results reveal a previously undescribed function of Cavin-Caveolin system in the development of PAH through regulation of BMP/Smad signaling. Funding Acknowledgement Type of funding source: None

Endothelial Notch signaling controls insulin transport in muscle

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ‐specific manner. Insulin flux across the endothelium to muscle cells is a rate‐limiting process influencing insulin‐mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non‐obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC‐specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high‐fat diet‐induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

Caveolins; An Assailant or An Ally of Various Cellular Disorders

Caveolae have impressive morphological highlights of the cytomembrane of mammalian cells which involve in wide diversity of cellular functions involving signaling pathways and cholesterol hastening. Caveolin proteins possess a 'scaffolding' domain which for caveolin-1 and caveolin-3 appear to act a dominant role in signal regulation through caveolae. Caveolin-1 is treated to be protein in the cytomembrane entrapped with caveolae in endothelial cells and vascular smooth muscle cells which diminish nitric oxide (NO) by fill up the calcium/calmodulin (Ca2+/CaM) confining point of endothelial nitric oxide synthase (eNOS), decrease NO generation produce endothelial dysfunction and atherosclerotic injury development. It is a cholesterol-binding layer protein associated with cell cholesterol transport and also shows cardioprotective action through ischemic preconditioning (IPC) in diabetic and postmenopausal rat heart. Additionally it is ensnared in the procedures of tumorigenesis, prostate disease, and inflammation. The present study in the paper is to explore the structural functionalities of caveolins and their contributory role in CVS disorders and various other diseases.

Autotaxin and vascular endothelial growth factor receptor-2 and -3 are related to vascular development during the progression of chronic viral hepatitis C.

Vascular endothelial growth factor (VEGF) and autotaxin (ATX) play important roles in embryonic vasculogenesis and cancer progression. This study examines whether these two angiogenic factors cooperate in the mechanism that regulates vascular development during the progression of chronic viral hepatitis C (CVH-C) (Inuyama classification, F1-F4). First, surgical wedge biopsy specimens and needle biopsy specimens were obtained. Immunohistochemical staining for ATX and vascular endothelial growth factor receptor was assessed in serial sections. Immunoelectron microscopy was conducted with a perfusion-fixation method. In normal control liver tissue specimens, ATX was expressed at low levels within the branches of the hepatic artery and hepatic sinusoids. In F1 CVH-C liver tissue specimens, ATX was expressed within the branches of the hepatic artery. Additionally, VEGFR-2 was expressed within the branches of the hepatic artery and capillaries. In F3-F4 CVH-C liver tissue specimens, positive staining for ATX and VEGFR-2 or VEGFR-3 was detected in the branches of the hepatic artery or microlymphatic vessels. ATX-1 reaction products were specifically expressed on the plasma membrane of some microvascular endothelial cells (ECs) in the proliferative capillary artery. VEGFR-2 was expressed on caveolae in ECs and vascular smooth muscle cells. VEGFR-3 immunogold particles were also observed in lymphatic ECs. These results suggest functional interactions among ATX, VEGFR-2, and VEGFR-3 in the modulation of hemovascular and lymphovascular cell activation during vascular development.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+]i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Intracellular transcytosis of albumin in glomerular endothelial cells after endocytosis through caveolae.

We previously described albumin endocytosis through caveolae in human renal glomerular endothelial cells (HRGECs). This suggested a new albumin transcytosis pathway, in addition to the fenestral pathway. As a next step, we investigated albumin transcytosis in HRGECs after caveolar endocytosis. HRGECs were incubated with Alexa Fluor 488-labeled bovine serum albumin from 0 to 360 min. Next, markers for endosomes, endoplasmic reticulum (ER), golgi apparatus (GA), lysosomes, and proteasomes and Fc receptors, microtubules, and actin were monitored by immunofluorescence. Labeled albumin co-localization with endosomes was gradually and significantly increased and it was significantly higher than with the other markers at any timepoint. Albumin, placed on inside of the Transwell membrane, diffused through HRGEC monolayers during a 360 min incubation period. This transportation of albumin through HRGECs was inhibited by methyl beta cyclodextrin (MBCD), a caveolae disrupting agent. MBCD also decreased albuminuria, causing decreased caveolin-1 (Cav-1) expression on glomerular capillaries, in puromycin aminonucleoside induced nephrotic mice. Albumin transcytosis depends on early endosomes, but not on other organelles, Fc receptors, or cytoskeletal components. Caveolae disruption prevented albumin transportation through HRGECs and decreased albuminuria in nephrotic mice. This newly described caveolae-dependent albumin pathway through glomerular endothelial cells is a potential pathogenetic mechanism for albuminuria, independent of the fenestrae.

Cavin‐3 deficiency reduces caveolae abundance in vascular and urinary bladder smooth muscle cells

We investigated the effect of cavin‐3 (protein kinase C delta binding protein, Prkcdbp) deficiency on vascular and urinary bladder function. Small mesenteric arteries and the urinary bladder were isolated from mice lacking cavin‐3 (Prkcdbp−/−, knockout: KO) and from control littermates (Prkcdbp+/+, wild type: WT). One group of mice was used for blood pressure measurements, whereas another set of mice underwent bladder outlet obstruction followed by analysis of protein expression using western blotting. Our studies revealed that loss of cavin‐3 resulted in a 40% reduction of the caveola protein cavin‐1 (polymerase I and transcript release factor, Ptrf) in vascular and bladder smooth muscle. Electron microscopy demonstrated that loss of cavin‐3 was accompanied by a reduction of caveolae by 40% in smooth muscle, while the abundance of caveolae in endothelial cells was unchanged. Vascular contraction in response to cirazoline (α1‐adrenergic agonist) was normal, but nitric‐oxide dependent relaxation was enhanced. The latter effect was associated with elevated expression of soluble guanylyl cyclase, and yet blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by loss of cavin‐3, and the proteomic response to outlet obstruction, including STAT3 (signal transducer and activator of transcription 3) phosphorylation, induction of synthetic markers, and repression of contractile markers were identical, the only exception being a curtailed induction of the Golgi protein GM130 (Golgi matrix protein 130 kD). In conclusion, loss of cavin‐3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin‐1, but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and a somewhat disturbed Golgi homeostasis in a situation of severe cellular stress.Support or Funding InformationThis study was funded by The Swedish Research Council (Vetenskapsrådet, http://www.vr.se/, K2015‐65X‐22662‐01‐3/KS), the Crafoord Foundation (http://en.crafoord.se/, CR), and the Royal Physiographic Society (Kungliga Fysiografiska Sällskapet, http://www.fysiografen.se/, CR).

Helium Influences Caveolae Numbers in Left Ventricular Endothelial Cells and Alters Metabolic Profile in Mice

BackgroundMyocardial ischemia and reperfusion (I/R) injury is a major cause of morbidity and mortality. IR injury leads to calcium overload, increased oxidative stress, mitochondrial dysfunction and ultimately apoptosis. Helium, an inert gas, causes cardioprotection potentially through secretion of cellular factors into serum. This study aimed to elucidate the protective potential of helium conditioned serum, the metabolite content of serum, and the impact of helium on endothelial cell ultrastructure. We hypothesized that helium induces the release of circulating factors to mediate conditioning and impacts endothelial cell ultrastructure.Materials and methodsMice were exposed to 70% helium/30% oxygen or 30% oxygen in air. Serum was harvested at 30 min, 4 hrs and 24 hrs after gas exposure (n=7/group). In addition, mice (n=4/group) were prepared and fixed for transmission electron microscopy. High magnification images (11000×) of left ventricular tissue were analyzed for the amount of caveolae in endothelial cells and myocytes. Serum from exposed mice was analyzed for metabolites of primary metabolism and complex lipids by mass spectrometry. Metabolites were categorized by physiologic pathways and multivariate analysis was used to determine interaction between helium and recover time.ResultsEndothelial cell caveolae number was increased at 30 min and 24 hrs post exposure in the helium group by 39% (p < 0.01, C: 3.5 ± 0.2, n=83 images; He: 4.8 ± 0.3 caveolae/mm, n=67 as mean ± SEM) and 18% (p < 0.05, C: 4.3 ± 0.3, n=72; He: 5.3 ± 0.3 caveolae/mm, n=61), respectively. No differences were observed in myocyte caveolae formation. Interaction of time and drug showed a trend for oxidative phosphorylation (p= 0.097) and galactose metabolism pathway (p= 0.0798). An overall drug effect between the different time points was observed for biotin metabolism (p < 0.05). The metabolites of the two pathways, valine/leucine/isoleucine and pantothenate/CoA biosynthesis showed a trend towards a drug effect (p=0.053; p=0.057 respectively). Three pathways were significantly changed in the 4 hrs group and in the 24 hrs group biosynthesis of unsaturated fatty acids was influenced. In addition three cardioprotective ceramides were significantly upregulated by helium (p < 0.05).ConclusionHelium exposure altered endothelial cell ultrastructure by inducing the formation of caveolae and induced secretion of factors resulting in different metabolic profiles of primary metabolism and complex lipids. Such changes in cell ultrastructure and metabolite secretion may be key to the conditioning effects induced by helium.

Cavin-3 (PRKCDBP) deficiency reduces the density of caveolae in smooth muscle

Cavins belong to a family of proteins that contribute to the formation of caveolae, which are membrane organelles with functional roles in muscle and fat. Here, we investigate the effect of cavin-3 ablation on vascular and urinary bladder structure and function. Arteries and urinary bladders from mice lacking cavin-3 (knockout: KO) and from controls (wild type: WT) were examined. Our studies revealed that the loss of cavin-3 resulted in ∼40% reduction of the caveolae protein cavin-1 in vascular and bladder smooth muscle. Electron microscopy demonstrated that the loss of cavin-3 was accompanied by a reduction of caveolae abundance by 40-45% in smooth muscle, whereas the density of caveolae in endothelial cells was unchanged. Vascular contraction in response to an α1-adrenergic agonist was normal but nitric-oxide-dependent relaxation was enhanced, in parallel with an increased relaxation on direct activation of soluble guanylyl cyclase (sGC). This was associated with an elevated expression of sGC, although blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by the loss of cavin-3. The proteomic response to outlet obstruction, including STAT3 phosphorylation, the induction of synthetic markers and the repression of contractile markers were identical in WT and KO mice, the only exception being a curtailed induction of the Golgi protein GM130. Loss of cavin-3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin-1 but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and slightly disturbed Golgi homeostasis in situations of severe cellular stress.

Influence of Surface Modifications on the Spatiotemporal Microdistribution of Quantum Dots In Vivo.

For biomedical applications of nanoconstructs, it is a general prerequisite to efficiently reach the desired target site. In this regard, it is crucial to determine the spatiotemporal distribution of nanomaterials at the microscopic tissue level. Therefore, the effect of different surface modifications on the distribution of microinjected quantum dots (QDs) in mouse skeletal muscle tissue has been investigated. In vivo real-time fluorescence microscopy and particle tracking reveal that carboxyl QDs preferentially attach to components of the extracellular matrix (ECM), whereas QDs coated with polyethylene glycol (PEG) show little interaction with tissue constituents. Transmission electron microscopy elucidates that carboxyl QDs adhere to collagen fibers as well as basement membranes, a type of ECM located on the basolateral side of blood vessel walls. Moreover, carboxyl QDs have been found in endothelial junctions as well as in caveolae of endothelial cells, enabling them to translocate into the vessel lumen. The in vivo QD distribution is confirmed by in vitro experiments. The data suggest that ECM components act as a selective barrier depending on QD surface modification. For future biomedical applications, such as targeting of blood vessel walls, the findings of this study offer design criteria for nanoconstructs that meet the requirements of the respective application.

Effect of quercetin and its metabolite on caveolin-1 expression induced by oxidized LDL and lysophosphatidylcholine in endothelial cells.

Oxidized low-density lipoprotein contributes to atherosclerotic plaque formation, and quercetin is expected to exert anti-atherosclerotic effects. We previously reported accumulation of conjugated quercetin metabolites in the aorta of rabbits fed high-cholesterol diets with quercetin glucosides, resulting in attenuation of lipid peroxidation and inhibition of lipid accumulation. Caveolin-1, a major structural protein of caveolae in vascular endothelial cells, plays a role in atherosclerosis development. Here we investigated effects of oxidized low-density lipoprotein, quercetin and its metabolite, quercetin 3-O-β-glucuronide, on caveolin-1 expression. Oxidized low-density lipoprotein significantly upregulated caveolin-1 mRNA expression. An oxidized low-density lipoprotein component, lysophosphatidylcholine, also induced expression of both caveolin-1 mRNA and protein. However, lysophosphatidylcholine did not affect the location of caveolin-1 proteins within caveolae structures. Co-treatment with quercetin or quercetin 3-O-β-glucuronide inhibited lysophosphatidylcholine-induced caveolin-1 expression. Quercetin and quercetin 3-O-β-glucuronide also suppressed expression of adhesion molecules induced by oxidized low-density lipoprotein and lysophosphatidylcholine. These results strongly suggest lysophosphatidylcholine derived from oxidized low-density lipoprotein contributes to atherosclerotic events by upregulating caveolin-1 expression, resulting in induction of adhesion molecules. Quercetin metabolites are likely to exert an anti-atherosclerotic effect by attenuating caveolin-1 expression in endothelial cells.

Caveolin-1 in Cardiovascular Disease: A Double-Edged Sword.

Endothelial dysfunction, as manifested by an attenuation of nitric oxide (NO)–mediated vasodilation, is recognized to be a fundamental abnormality in the genesis of hypertension, atherosclerosis, and coronary artery disease (1). Metabolic risk factors, such as obesity, insulin resistance, and type 2 diabetes (T2D), can initiate and accelerate endothelial dysfunction leading to cardiovascular disease (CVD) (1). Endothelial dysfunction in association with metabolic abnormalities is typically caused by a combination of reduced production and increased destruction of NO leading to a decrease in NO bioavailability (1,2). NO production in response to various factors, such as increased shear stress, is mediated by endothelial nitric oxide synthase (eNOS), which is constitutively expressed in endothelial cells (ECs) and is tightly controlled by various membrane-bound receptors and regulatory proteins under physiological conditions (3). Caveolin-1 (Cav-1), an anchoring protein in the plasma membrane caveolae in ECs and vascular smooth muscle cells (VSMCs), attenuates endothelial NO production by occupying the calcium/calmodulin (Ca2+/CaM) binding site of eNOS (4) (Fig. 1). Increases in caveolin and eNOS interaction, as may occur with hyperlipidemia, reduce NO production and promote endothelial dysfunction and atherosclerotic lesion formation. This process is mediated by increased lipoprotein trafficking across the vascular endothelium (5,6). Therefore, treating hyperlipidemia as an early intervention to help …

Caveolae protect endothelial cells from membrane rupture during increased cardiac output

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.

Evidence for functional and dynamic microcompartmentation of Cav-1/TRPV4/KCa in caveolae of endothelial cells

Ca2+-activated K+ channels (KCa) play a pivotal role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, KCa depend on an increase of intracellular calcium which is substantially mediated by Ca2+-permeable cation channels including the transient receptor potential V4 (TRPV4). It has been proposed that KCa and Ca2+-permeable cation channels may be clustered in localized positions within the cell membrane to form functional units and that caveolae may constitute the scaffolding for such microcompartmental organization. Here, we sought to elucidate the composition and functional relevance of these microcompartments in vitro and in vivo. We show that TRPV4 and small-conductance KCa2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found a caveolae-dependent association between caveolin-1, TRPV4 and small conductance KCa2.3, but not intermediate conductance KCa3.1, in endothelial cells under static condition. Mechanical stimulation of cells via exposure to shear stress led to a partial de-novo colocalization of KCa3.1 with Cav-1 and TRPV4. In a mouse model of genetic Cav-1 deficiency, we found significantly reduced KCa-mediated currents as determined by patch-clamping in carotid artery endothelial cells (CAEC) from Cav-1−/− mice compared to wildtype. Functionally, Cav-1 deficiency was associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in response to shear stress and acetylcholine. In summary, our findings provide evidence for a dynamic microcompartmentation of TRPV4/KCa in caveolae of endothelial cells and highlight the importance of Cav-1 for endothelial KCa functions and flow-induced vasodilation.

Exploring GPCR Biased Signaling from inside and outside the Cell

G protein‐coupled receptors (GPCRs) are the largest family of cell surface receptors expressed in the mammalian genome and mediate vast physiological responses, making this receptor class the target of most drugs used clinically. GPCRs are dynamic molecules that assume multiple active states that bias signaling towards distinct G‐protein subtypes and beta‐arrestins. However, the mechanisms that regulate GPCR bias signaling remains unclear. Towards understanding the molecular basis of GPCR biased signaling, we examined the role of post‐translational modifications and plasma membrane compartmentalization. We discovered that N‐linked glycosylation of protease‐activated receptor‐1 (PAR1), a GPCR for the coagulant protease thrombin, regulates preferential coupling to specific G‐protein subtypes that control stress fiber formation and cellular proliferation. We also found that PAR1 distribution into caveolae in endothelial cells is critical for biased signaling induced by thrombin versus activated Protein C, an anti‐coagulant protease. Thrombin‐activated PAR1 coupled to G‐proteins and induced endothelial barrier permeability. In contrast, APC‐activated PAR1 signaled preferentially through beta‐arrestins to promote endothelial barrier stabilization, which required compartmentalization in caveolae. These studies reveal two different mechanisms that regulate GPCR biased signaling from inside and outside of the cell. 
 In addition to her research, Dr. JoAnn Trejo is an experienced educator and leader of diversity efforts. She will also discuss the untapped potential: diversity and the advancement of science.

Increases in endothelial caveolin-1 and cavins correlate with cirrhosis progression

Background and aimsCaveolin-1 is associated with flat caveolar domains, invaginated smooth plasmalemmal vesicles, and caveolae. Polymerase 1 and transcript release factor (PTRF) (cavin 1) and serum deprivation protein response (SDPR) (cavin 2) are required for the invagination of caveolae, and PRKCDBP (protein kinase C, delta-binding protein; cavin 3) is required for caveolae budding to form caveolar vesicles. To investigate whether cavins are involved in hepatic sinusoidal angiogenesis and remodeling during progression to cirrhosis, normal control liver specimens and early and late cirrhotic liver specimens were studied. Materials and methodsCavin-1, cavin-2, and cavin-3 proteins and their gene expression were examined using immunohistochemistry (IHC), Western blotting, and laser capture microdissection (LCM)-polymerase chain reaction (PCR) during progression of cirrhosis caused by hepatitis C. According to the perfusion, fixation methods were designed to reevaluate the precise ultrastructural localizations and changes of cavin-1 and cavin-2 expression on liver sinusoidal endothelial cells (LSECs) facing the sinusoidal blood flow. ResultsFor IHC, cavin-1 and cavin-2 expressions were found to be upregulated in small angiogenic LSECs with collagen deposition in the perisinusoidal space as well as in the vascular endothelial cells of the remarkably proliferated portal venules, hepatic arterioles, and arterial capillaries within the fibrotic septa of late-stage cirrhotic liver. Cavin-3 was mainly localized in large vessels, and it was detected only scantly on the central vein and hepatic sinusoids in the control liver. In late-stage cirrhotic liver, the intensity of cavin-3 was enhanced mainly on proliferative large vessels in regenerated nodules and in the peripheral regions of nodules and fibrous septa. On conducting immunoelectron microscopy, in the control liver tissue, cavin-1 was found to be localized on the caveolae of hepatic arterial and portal venous endothelial cells, but it was scantly localized on hepatic sinusoidal lining cells, and cavin-2 was found mainly on vesicles in LSECs. In the cirrhotic liver tissue, aberrant cavin-1 and cavin-2 expressions were observed on caveolae-like structures in LSECs. Significant overexpressions of cavin-1 at the protein and messenger RNA (mRNA) levels in a cirrhotic liver were demonstrated by Western blotting and LCM-PCR. ConclusionsCavin-1 and cavin-2 are strongly expressed within caveolae-like structures and associated vesicles within LSECs of the hepatitis C-related cirrhotic liver. Cavin-1 would play a critical role in regulating aspects of caveolin-1 in LSECs. Moreover, these findings suggest a direct association of cavin-1 and cavin-2 with the process of differentiation and transformation of LSECs inducing hepatic sinusoidal capillarization related to the progression of cirrhosis.