Cav-1 Levels Research Articles

Helium alters the cytoskeleton and decreases permeability in endothelial cells cultured in vitro through a pathway involving Caveolin-1

Caveolins are involved in anaesthetic-induced cardioprotection. Actin filaments are located in close connection to Caveolins in the plasma membrane. We hypothesised that helium might affect the cytoskeleton and induce secretion of Caveolin. HCAEC, HUVEC and Cav-1 siRNA transfected HUVEC were exposed for 20 minutes to either helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2). Cells and supernatants were collected for infrared Western blot analysis, immunofluorescence staining, nanoparticle tracking analysis and permeability measurements. Helium treatment increased the cortical localisation of F-actin fibers in HUVEC. After 6 hours, helium decreased cellular Caveolin-1 (Cav-1) levels and increased Cav-1 levels in the supernatant. Cell permeability was decreased 6 and 12 hours after helium treatment, and increased levels of Vascular Endothelial - Cadherin (VE-Cadherin) and Connexin 43 (Cx43) were observed. Transfection with Cav-1 siRNA abolished the effects of helium treatment on VE-Cadherin, Cx43 levels and permeability. Supernatant obtained after helium treatment reduced cellular permeability in remote HUVEC, indicating that increased levels of Cav-1 are responsible for the observed alterations. These findings suggest that Cav-1 is secreted after helium exposure in vitro, altering the cytoskeleton and increasing VE-Cadherin and Cx43 expression resulting in decreased permeability in HUVEC.

Reciprocal regulation of the Cadherin-11/Stat3 axis by caveolin-1 in mouse fibroblasts and lung carcinoma cells

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.

TGF-β1 Regulation of P-JNK and L-Type Calcium Channel Cav1.2 in Cortical Neurons.

Central nervous system (CNS) diseases can cause a series of neuronal lesions, which may be improved by the anti-apoptotic neuroprotection of transforming growth factor-beta 1 (TGF-β1). In neurons, L-type Ca2+ channels (LTCC) are mainly composed of Cav1.2 subunits. Given the implication of TGF-β1 in numerous CNS diseases, we examined the neuroprotective effects of TGF-β1 on the Cav1.2 channel in the CNS. To simulate acute mechanical traumatic brain injury (TBI), we used a needle to create parallel scratches across plates, which were cultured for 9h. Meanwhile, Fluo4-AM-loaded laser scanning confocal microscopy with a dual wavelength of 488nm/530nm was employed to determine intracellular calcium concentrations ([Ca2+]i). We found that MAPK inhibitors impede TGF-β1-induced cell viability and that TGF-β1 recovered from the trauma-induced cell viability in neurons. Cav1.2 production was significantly decreased in the TGF-β1-treated (10ng/mL) neurons. At this TGF-β1 concentration, Cav1.2 was significantly down-regulated in a time-dependent manner after 12h. Moreover, TGF-β1 partially recovered the protein levels of Cav1.2 that were reduced by TBI. TGF-β1 significantly inhibited the fluorescence intensity of [Ca2+]i increased by KCl and delayed the time of the peak [Ca2+]i. The observed effects of TGF-β1 on Cav1.2 were regulated by MAPK inhibitors. The observed effects of TGF-β1 on P-JNK were also impeded by pre-incubation with the LTCC inhibitor (10μM) nimodipine in trauma-injured neurons. Altogether, TGF-β1 regulated LTCCs through a mechanism dependent on MEK, JNK1/2 and p38 MAPK signal pathways in cortical neurons. Thus, we suggest the involvement of this mechanism in cell viability.

Expression of eNOS and CAV gene in trabecular meshwork cells and effects on glaucoma pathogenesis

Objective: Glaucoma is the second popular blinding disease worldwide, and is mainly caused by abnormal function or structure of trabecular meshwork and consequent aqueous humor effluent dysfunction, ocular hypertension, leading to vision deficit. eNOS and CAV1 might be correlated with glaucoma onset. This study thus investigated their expression in trabecular meshwork cells and effects on glaucoma pathogenesis. Patients and Methods: Trabecular meshwork tissues were collected during surgery from POAG patients, and eNOS/CAV1 gene expressions were analyzed by qRT-PCR and Western-Blot. eNOS and CAV1 genes were over-expressed in cultured HTMC cells, whose survival rate was analyzed by MTT assay, whilst cell free radicals clearance rate was analyzed by chemiluminescence method. Results: HE staining revealed significant pigment deposition in trabecular meshwork of POAG patients compared to normal group, accompanied with stenosis of mesh. Expression levels of eNOS and CAV1 were significantly higher in POAG group (p<0.05 compared to healthy control group). In vitro overexpression of eNOS and CAV1 significantly suppressed cell survival rate and free radical clearance rate (p<0.05). Conclusion: Trabecular meshwork cells in glaucoma patients showed elevated eNOS and CAV1 expression. Up-regulation of eNOS expression may suppress cell survival rate at cellular level, impaired ability to clear free radicals, eventually leading to cell or tissue damage.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Caveolin-1 regulates autophagy activity in thyroid follicular cells and is involved in Hashimoto's thyroiditis disease.

Hashimoto's thyroiditis (HT) is considered a T helper-type 1 (Th1) cytokine-dominant autoimmune thyroid disease. Caveolin-1 (Cav-1), a part of the thyroxisome multiprotein complex, is localized at the apical pole of thyrocytes and is indispensable for synthesis of thyroid hormones and modulation of oxidative stress in order to avoid cell damage and apoptosis. Reduced autophagy induces thyroid follicular cells (TFC) apoptosis by activating reactive oxygen species (ROS) in HT patients. Nevertheless, whether Cav-1 has roles in the regulation of autophagy remains largely unclear. In this study, we examined Th1 cytokines and Cav-1 expression in HT thyroid tissues, determined the effects of interleukin-1beta (IL-1β) and interferon-gamma (IFN-γ) on Cav-1 and autophagy activity in TFC, and investigated the association between Cav-1 and autophagy activity in vitro. Our results indicate that higher levels of IL-1β and IFN-γ and lower levels of Cav-1 were expressed in thyroid tissues of HT patients than in those of normal controls. Cav-1 mRNA and protein levels were significantly decreased in TFC exposed to IL-1β and IFN-γ, accompanied by decreased expression of autophagy-related protein LC3B-II. Interestingly, small interfering RNA (siRNA)-mediated Cav-1 knockdown in TFC reduced LC3B-II protein expression. Taken together, these results suggest that lack of Cav-1 expression inhibited autophagy activity in TFC exposed to Th1 cytokines (IL-1β and IFN-γ), which might be a novel pathogenetic mechanism of HT.

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1);...

Loss of Caveolin-1 in Metastasis-Associated Macrophages Drives Lung Metastatic Growth through Increased Angiogenesis

SummaryAlthough it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1).

The role of caveolin-1 in morphine-induced structural plasticity in primary cultured mouse cerebral cortical neurons.

Long-term repeated application of morphine induces plasticity changes in sensitive neurons, which represents the neurobiological basis of morphine addiction. Caveolin-1 (Cav-1) is an important scaffolding protein that plays a key role in the regulation of neurostructural plasticity. However, in-depth studies focused on the role of Cav-1 in morphine-induced changes in neurostructural plasticity are lacking. The present study explored the effects of Cav-1 on the expression levels of 2 markers of neurite outgrowth, growth association protein 43 (GAP-43) and microtubule-associated protein 2 (MAP-2), during the process of morphine-induced changes in the structural plasticity. A primary mouse cerebral cortical neuron culture was established, and Cav-1 knockdown was achieved. The results showed that morphine at a concentration of 10.0μmol/L had no adverse effect on neuronal viability, but enhanced the Cav-1 and GAP-43 levels and induced the outgrowth of MAP-2-labeled neurites. The qRT-PCR data showed that 10.0μmol/L of morphine increased the Cav-1 mRNA level and promoted Cav-1 expression at the transcriptional level. Moreover, Cav-1 knockdown inhibited the morphine-induced upregulation of GAP-43 expression and the prolongation of MAP-2-labeled neurites. In conclusion, morphine was capable of inducing changes in the structural plasticity of primary cultured mouse cortical neurons, and Cav-1 played an important role in this process. Inhibition of Cav-1 expression reduced the morphine-induced increase in the neuronal growth markers GAP-43 and MAP-2. Thus, Cav-1 may be a potential molecular target for inhibiting morphine-induced neuroplasticity.

Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and \u03b21 integrin

Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that β1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated β1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-β-cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and β1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phospho-mimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in β1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering β1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between β1 integrin and Cav1. Finally, co-expression of this auto-clustering β1 integrin and Cav1-Y14D synergistically enhanced cell spreading, and FA assembly in HEK293T cells cultured on either stiff ( > G Pa) or soft (0.2 kPa) matrices. Collectively, these results suggest that matrix stiffness governs the expression of β1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.

Roles and mechanisms of TRPC3 and the PLCγ/PKC/CPI-17 signaling pathway in regulating parturition.

The aim of the current study was to investigate the role of phospholipase C (PLC)γ/protein kinase C (PKC)/C-kinase-activated protein phosphatase-1 (CPI-17) signaling pathways in uterine smooth muscle during parturition. Samples of uterine tissue were collected from pregnant patients who underwent a caesarean section for preterm delivery, full-term delivery with labor onset, full-term delivery without labor onset, and from a non-pregnant control group undergoing surgery for cervical intraepithelial neoplasia III. Immunohistochemistry, and western blotting were used to assess the association between TRPC3 levels and parturition and the influence of calcium ion channels. In addition, pregnant mice were used to explore the effect of uterine canonical transient receptor potential 3 (TRPC3) expression on the parturition-triggering mechanism and PLCγ/PKC/CPI-17 signaling pathways. Pregnant mouse uterine smooth muscle cells were cultivated, with and without TRPC3 silencing, and the expression levels of PLCγ, PKC and CPI-17, the upstream and downstream factors of the TRPC3 pathway, were measured in pregnant mouse uterine smooth muscle cells, in order to provide a theoretical basis for the prevention and treatment of premature labor. In the preterm and full-term without labor onset patient groups, the TRPC3 gene expression in the mSMCs was significantly overexpressed when compared with the non-pregnant group (P<0.05); however, TRPC3 expression was not elevated in the full-term with labor onset group, exhibiting no significant difference compared with the non-pregnant group (P>0.05). During pregnancy, compared with the non-pregnant controls, Cav1.2, Cav3.1 and Cav3.2 gene expression levels were markedly increased (P<0.05) in mSMCs from the preterm delivery group and the full-term with labor onset group, however were non-significantly increased in the full-term without labor onset group. The level of TRPC3 was highest in the preterm group, while the levels of Cav1.2, Cav3.1 and Cav3.2 were highest in the full-term with labor onset group. In the preterm, LPS-treated preterm and full-term groups, TRPC3, MAPK, ERK1/2, P-ERK, Cav3.2, Cav3.1 and Cav1.2 were all expressed at higher levels than in the unfertilized group. In the LPS-treated preterm group, the levels of TRPC3, MAPK, ERK1/2, P-ERK, Cav3.2, Cav3.1 and Cav1.2 were increased compared with the preterm group. Furthermore, following transfection of small interfering TRPC3 (siTRPC3) into cells, it was demonstrated that the levels of TRPC3, PLCγ, PKC, CPI-17, P-CPI-17, Cav1.2, Cav3.1 and Cav3.2 expression were lower in the LPS siTRPC3 group when compared with that of the LPS-treated untransfected control group.

Formaldehyde regulates vascular tensions through nitric oxide-cGMP signaling pathway and ion channels

Formaldehyde (FA) has been linked to the detrimental cardiovascular effects. Here, we explored the effects and mechanisms of FA on rat aortas both in vivo and in vitro. The results presented that FA evidently lowered the blood pressures of rats. The expression levels of BKCa subunits α and β1 and iNOS of the aortas were up-regulated by FA in vivo. However, FA markedly reduced the levels of Cav1.2 and Cav1.3, which are the subunits of L-Ca2+ channel. Furthermore, the contents of NO, cGMP and iNOS in the aortas were augmented by FA. To further confirm these findings, the mechanisms accredited to these effects were examined in vitro. The data showed that FA contracted the isolated aortic rings at low concentrations (<300 μM), while it relaxed the rings at high concentrations (>500 μM). The FA-induced vasoconstriction at low concentrations was blocked partly by an inhibitor of ACE. The relaxation caused by FA at high concentrations was attenuated by the inhibitors of NO-cGMP pathway, L-Ca2+ channel and BKCa channel, respectively. Similarly, the expression of iNOS was strongly enhanced by FA in vitro. The effects of FA on the aortic rings with endothelium were significantly greater than those on the rings without endothelium. Our results indicate that the vasoconstriction of FA at low concentrations might be partially pertinent to endothelin, and the FA-caused vasorelaxation at high concentrations is possibly associated with the NO-cGMP pathway, L-Ca2+ channel and BKCa channel. This study will improve our understanding of the pathogenic mechanisms for FA-related cardiovascular diseases.

The diversity in the expression profile of caveolin II transcripts, considering its new transcript in breast cancer.

Most studies have revealed the effects of caveolins in cancer inhibition. However, due to a lack of reports about their new transcripts, their presence and their effects on different cancers are unclear. This study was conducted to evaluate the cavolin-2 (cav-2) transcripts expression changes in tumoral and corresponding tissues and in contralateral breast, to investigate their variation associated with the variation of caveolin-1 (cav-1) expression in breast cancer. There were 40 breast-derived tumoral, corresponding, and contralateral tissues obtained from the patients with breast cancer. The RNA and proteins were extracted from these samples. So, cav-1 and cav-2 transcripts' variation were assessed in whole tumoral, corresponding, and contralateral breast. Also, their expression modifications were evaluated via the Western blotting technique. The results derived from this study verified the presence of transcript III of cav-2 for the first time, which was reported only in the gene bank, but we could not detect and validate any protein associated with these transcripts. Also, the decreasing trend of cav-1 and the cav-2 (transcripts I and II) were observed in tumoral tissues compared to unaffected tissues especially in stages I and II. It seems that the descending expression levels of cav-1 and cav-2 (transcript I, II) besides the lasting expression of cav-2 (transcript III) are associated with the incidence and promotion of breast cancer, especially in the initial stages of breast cancer. So, this may show a potential in determining the patients who can undergo the prophylactic mastectomy. Moreover, the results of the study demonstrated that transcript III may be a candidate as a non-coding RNA.

Effect of Low-Intensity Pulsed Ultrasound on the Expression of Calcium Ion Transport-Related Proteins during Tertiary Dentin Formation

Low-intensity pulsed ultrasound (LIPUS) is known for its positive effect on bone healing and reparative regeneration. This study investigated whether LIPUS affects reparative progression of the tooth and the expression of calcium ion transport-related proteins in odontoblasts and dental pulp cells using a rat dentin–pulp complex injury model. Forty male adult Sprague-Dawley rats underwent cavity preparation in the right maxillary first molar: 20 received LIPUS irradiation on the cavity-prepared tooth; 20 received LIPUS irradiation on the left maxillary first molar. Rats were randomly allocated into four groups: blank control group, LIPUS group, cavity-prepared group, cavity-prepared + LIPUS group. LIPUS irradiation (frequency: 1.5 MHz, 200-µs pulse width, 1-kHz pulse repetition frequency, 30 mW/cm2 spatial averaged temporal averaged intensity) was administered individually for 20 min daily. Rats were sacrificed 1, 3, 7 and 14 d post-operation. The histopathological and cellular morphologic changes in the dentin–pulp complex were detected with hematoxylin and eosin staining. Expression of calcium ion transport-related proteins (Cav1.2, NCX1 and TRPV1) was determined with immunohistochemical staining and imaging analysis. Histopathological analysis revealed obvious reparative dentin formation at day 14 in the cavity-prepared + LIPUS group compared with the other groups. Expression levels of Cav1.2, NCX1 and TRPV1 increased significantly by 22%, 53% and 23%, respectively, at day 1 and increased significantly by 23%, 27% and 22%, respectively, at day 3 in the cavity-prepared + LIPUS group (p < 0.05) compared with the cavity-prepared group. LIPUS has a positive effect on the expression of calcium transport-related proteins during early-stage dentin injury and facilitates tertiary dentin formation; the mechanism for this likely relates to the inflammatory reaction and a mechanical effect.

Baseline and longitudinal plasma caveolin-1 level as a biomarker in active surveillance for early-stage prostate cancer.

To evaluate the role of caveolin-1 (Cav-1) as a predictor of disease reclassification (DR) in men with early prostate cancer undergoing active surveillance (AS). We analysed archived plasma samples prospectively collected from patients with early prostate cancer in a single-institution AS study. Of 825 patients enrolled, 542 had ≥1 year of follow-up. Baseline and longitudinal plasma Cav-1 levels were measured using an enzyme-linked immunosorbent assay. Tumour volume or Gleason grade increases were criteria for DR. Logistic regression analyses were used to assess associations between clinicopathological characteristics and reclassification risk. In 542 patients, 480 (88.6%) had stage cT1c disease, 542 (100.0%) had a median prostate-specific antigen level of 4.1ng/mL, and 531 (98.0%) had a median Cancer of the Prostate Risk Assessment score of 1. In all, 473 (87.3%) had a Gleason score of 3+3. After a median of 3.1years of follow-up, disease was reclassified in 163 patients (30.1%). The meanbaseline Cav-1 level was 2.2 ± 8.5ng/mL and the median 0.2 ng/mL (range, 0-85.5 ng/mL). In univariate analysis, baseline Cav-1 was a significant predictor for risk ofDR (odds ratio [OR] 1.82, 95% confidence interval [CI] 1.24-2.65; P = 0.002). In multivariate analysis, with adjustments for age, tumour length, group risk stratification and number of positive cores, reclassification risk associated with Cav-1 remained significant (OR 1.91, 95% CI 1.28-2.84; P = 0.001). Baseline plasma Cav-1 level was an independent predictor of disease classification. New methods for refining AS and intervention may result.

Caveolin-1 expression as a prognostic marker in triple negative breast cancers of Asian women

BackgroundTriple-negative breast cancers (TNBCs) are defined by their lack of oestrogen receptor, progesterone receptor and epidermal growth factor receptor 2. Although heterogeneous, the majority are aggressive and treatment options are...

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.

Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.

Abstract 301: Prognostic value of metastatic tumoral caveolin-1 expression in patients with resected gastric cancer

Abstract Background : Caveolin-1 (Cav-1), as the main component of caveolae, has complex roles in tumourigenesis and clinical outcomes in a variety of human malignancies. We investigated Cav-1 in primary and metastatic tumor of gastric cancer (GC) and its association with clinical outcomes. Method : All specimens evaluated were obtained from 145 patients with GC who had undergone curative gastrectomy. The primary outcomes measured were the expression levels of Cav-1 by immunohistochemistry and its association with clinicopathologicalparameters and patient survival. Results : Among the 145 patients, the frequency of high expression of stromal Cav-1 within primary tumor and tumoral Cav-1 protein in metastatic lymph node were 22.5% (18/145) and 16.6% (15/91). In the multivariate analysis tumoral Cav-1 protein in metastatic lymph node showed prognostic significance for relapse-free survival (RFS, HR, 3.934; 95% CI, 1.882-8.224; P&amp;lt;0.001) and cancer-specific survival outcome (CSS, HR, 3.985; 95% CI, 1.620 - 8.623; P = 0.002). In addition, it remained as a strong indicator of poor prognosis for RFS (HR, 3.137; 95% CI, 1.445 - 6.810; P = 0.004) and CSS (HR, 2.509; 95% CI, 1.079 - 5.838; P = 0.033). Conclusions : High expression of tumoral Cav-1 protein in metastatic lymph node shows a strong negative association with the clinical outcomes in patients with curatively resected GC, suggesting that this protein have potential uses as novel prognostic markers. Citation Format: Jihyung Hong, Der Sheng Sun, Soon Auck Hong, Hye Sung Won, Han Hong Lee, Okran Kim, Yoon Ho Ko. Prognostic value of metastatic tumoral caveolin-1 expression in patients with resected gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 301. doi:10.1158/1538-7445.AM2017-301

Similar regulatory mechanisms of caveolins and cavins by myocardin family coactivators in arterial and bladder smooth muscle.

Caveolae are membrane invaginations present at high densities in muscle and fat. Recent work has demonstrated that myocardin family coactivators (MYOCD, MKL1), which are important for contractile differentiation and cell motility, increase caveolin (CAV1, CAV2, CAV3) and cavin (CAVIN1, CAVIN2, CAVIN3) transcription, but several aspects of this control mechanism remain to be investigated. Here, using promoter reporter assays we found that both MKL1/MRTF-A and MKL2/MRTF-B control caveolins and cavins via their proximal promoter sequences. Silencing of MKL1 and MKL2 in smooth muscle cells moreover reduced CAV1 and CAVIN1 mRNA levels by well over 50%, as did treatment with second generation inhibitors of MKL activity. GATA6, which modulates expression of smooth muscle-specific genes, reduced CAV1 and CAV2, whereas the cavins were unaffected or increased. Viral overexpression of MKL1 and myocardin induced caveolin and cavin expression in bladder smooth muscle cells from rats and humans and MYOCD correlated tightly with CAV1 and CAVIN1 in human bladder specimens. A recently described activator of MKL-driven transcription (ISX) failed to induce CAV1/CAVIN1 which may be due to an unusual transactivation mechanism. In all, these findings further support the view that myocardin family coactivators are important transcriptional drivers of caveolins and cavins in smooth muscle.

Abstract 266: Extracellular Hemoglobin From Stored Rbcs Binds Ago2 And Delivers Functional Microrna to Endothelial Cells

Red blood cell (RBC) transfusion is among the most common clinical therapies. Prior to transfusion, packed RBCs (PRBCs) may be stored for up to 42 days. During storage, RBCs undergo many changes, including hemolysis and the release of extracellular hemoglobin (Hb). Prior work has shown that endothelial cells exposed to free hemoglobin demonstrate upregulation of inflammatory and anti-inflammatory pathways including elevated ICAM-1, VCAM-1 and E selectin, as well as elevated Hemeoxygenase-1 and bilirubin levels. Our work tests the hypothesis that extracellular Hb from stored RBCs can transfer functional microRNA to endothelial cells. Stored RBCs (age 30-42 days) were obtained from the Emory University Hospital Blood Bank. Extracellular Hb was obtained from lysed RBCs. Binding of Argonaute 2 (Ago2) to Hb was assessed by immunoprecipitation (IP) of Hb-alpha followed by Western blot for Ago2. In addition, microRNA was extracted from the Hb immunoprecipitate. Levels of miR-451, -16, -584, and -92 were assessed by RT-qPCR analysis. Lastly, human aortic endothelial cells (HAECs) were treated with Hb from RBC lysates. After treatment, Qiazol was used to extract RNA from washed cells, and levels of miR-451 and target genes were measured by RT-qPCR and Western analysis. We found that Ago2, a crucial component of the RISC complex that executes the regulatory function of microRNA, co-immunopreciptated with Hb from RBC lysates. We also observed that miR-451, a microRNA known to be abundant in RBCs, was highly abundant in Hb immunoprecipates. Furthermore, miR-16, -584 and -92 could also be amplified from Hb immunoprecipitates. Endothelial cells treated with extracellular Hb from RBC lysates for 36 hours had a dramatic increase in levels of miR-451 and a significant decrease in mRNA abundance of genes known to be targets of miR-451, including ATF2 (p-value=0.03), CAV1 (p-value=0.02), and MIF (p-value=0.005). HAECs treated with extracellular Hb for 72 hours had decreased protein levels of ATF2 (p-value=0.01) and CAV1 (p-value=0.003). In conclusion, previous work has shown that extracellular Hb can have direct effects on endothelial cells; our data show, for the first time, that extracellular Hb also serves as a vehicle for the cell-to-cell delivery of microRNA.