Adipose stromal cells; Endothelial cells; Mural cell differentiation; Activin A
Vascular smooth muscle cells (VSMCs) undergo transcriptionally regulated reversible differentiation in growing and injured blood vessels. This de-differentiation also contributes to VSMC hyperplasia following vascular injury, including that caused by angioplasty and stenting. Stents provide mechanical support and can contain and release rapamycin, an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1). Rapamycin suppresses VSMC hyperplasia and promotes VSMC differentiation. We report that rapamycin-induced differentiation of VSMCs required the transcription factor GATA-6. Inhibition of mTORC1 stabilized GATA-6 and promoted the nuclear accumulation of GATA-6, its binding to DNA, and its transactivation of promoters encoding contractile proteins and inhibitors of proliferation. These effects were mediated by phosphorylation of GATA-6 at Ser290, potentially by Akt2, a kinase that is activated in VSMCs when mTORC1 is inhibited. Rapamycin induced phosphorylation of GATA-6 in wild-type mice, but not in Akt2−/− mice. Intimal hyperplasia after arterial injury was greater in Akt2−/− mice than in wild-type mice, and the exacerbated response in Akt2−/− mice was rescued to a greater extent by local overexpression of the wild-type or phosphomimetic (S290D) mutant GATA-6 than by that of the phosphorylation-deficient (S290A) mutant. Our data indicated that GATA-6 and Akt2 are involved in the mTORC1-mediated regulation of VSMC proliferation and differentiation. Identifying the downstream transcriptional targets of mTORC1 may provide cell type-specific drug targets to combat cardiovascular diseases associated with excessive proliferation of VSMCs.
Platelet abnormalities are well-recognized complications of diabetes mellitus (DM). Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in DM. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in DM platelets is unknown.
Methods and Results
Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase (AR) activation, and subsequent reactive oxygen species (ROS) production, leads to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage and rupture by sequestration of the anti-apoptotic protein Bcl-xL. In a glucose dose dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, AR activation, ROS production and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are two distinct states, dependent upon the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model.
AR contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for DM thrombotic complications.
aldose reductase; p53; platelet; diabetes mellitus; mitochondria; apoptosis
Smooth muscle cells (SMC) are remarkably plastic. Their reversible differentiation is required for growth and wound healing, but also contributes to pathologies including atherosclerosis and restenosis. While key regulators of the SMC phenotype including myocardin (MYOCD) and KLF4 have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported.
Methods and Results
Using human SMC, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its 5-hydroxymethylcytosine (5-hmC) product are enriched in contractile SMC but reduced in dedifferentiated SMC. TET2 knockdown inhibits expression of key pro-contractile genes including MYOCD and SRF with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, while TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regulates phenotypic modulation through opposing effects on chromatin accessibility at the promoters of pro-contractile versus dedifferentiation-associated genes. Notably, we find that TET2 binds, and 5-hmC is enriched, in CArG-rich regions of active SMC contractile promoters (MYOCD, SRF, and MYH11). Loss of TET2 and 5-hmC positively correlates with the degree of injury in murine models of vascular injury and human atherosclerotic disease. Importantly, localized TET2 knockdown exacerbates injury response while local TET2 overexpression restores the 5-hmC epigenetic landscape, contractile gene expression, and greatly attenuates intimal hyperplasia in vivo.
We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation.
smooth muscle; differentiation; gene expression/regulation; epigenetics; hyperplasia
Phenotypic plasticity in vascular smooth muscle cells (VSMC) is necessary for vessel maintenance, repair and adaptation to vascular changes associated with aging. De-differentiated VSMC contribute to pathologies including atherosclerosis and intimal hyperplasia. As resveratrol has been reported to have cardio- protective effects, we investigated its role in VSMC phenotypic modulation. We demonstrated the novel finding that resveratrol promoted VSMC differentiation as measured by contractile protein expression, contractile morphology and contraction in collagen gels. Resveratrol induced VSMC differentiation through stimulation of SirT1 and AMPK. We made the novel finding that low or high dose resveratrol had an initially different mechanism on induction of differentiation. We found that low dose resveratrol stimulated differentiation through SirT1-mediated activation of AKT, whereas high dose resveratrol stimulated differentiation through AMPK-mediated inhibition of the mTORC1 pathway, allowing activation of AKT. The health effects of resveratrol in cardiovascular diseases, cancer and longevity are an area of active research. We have demonstrated a supplemental avenue where-by resveratrol may promote health by maintaining and enhancing plasticity of the vasculature.
Supravalvular aortic stenosis (SVAS) is caused by mutations in the elastin (ELN) gene and is characterized by abnormal proliferation of vascular smooth muscle cells (SMCs) that can lead to narrowing or blockage of the ascending aorta and other arterial vessels. Availability of patient-specific SMCs may facilitate studying disease mechanisms and developing novel therapeutic interventions.
Methods and Results
Here, we report the development of a human induced pluripotent stem cell (iPSC) line from a patient with SVAS caused by the premature termination in exon 10 of the ELN gene due to an exon 9 4-nucleotide insertion. We showed that SVAS iPSC-derived SMCs (iPSC-SMCs) had significantly fewer organized networks of smooth muscle alpha actin (SM α-actin) filament bundles, a hallmark of mature contractile SMCs, compared to control iPSC-SMCs. Addition of elastin recombinant protein or enhancement of small GTPase RhoA signaling was able to rescue the formation of SM α-actin filament bundles in SVAS iPSC-SMCs. Cell counts and BrdU analysis revealed a significantly higher proliferation rate in SVAS iPSC-SMCs than control iPSC-SMCs. Furthermore, SVAS iPSC-SMCs migrated at a markedly higher rate to the chemotactic agent platelet-derived growth factor (PDGF) in comparison with the control iPSC-SMCs. We also provided evidence that elevated activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is required for hyper-proliferation of SVAS iPSC-SMCs. The phenotype was confirmed in iPSC-SMCs generated from a patient with deletion of elastin due to Williams-Beuren syndrome (WBS).
Thus, SVAS iPSC-SMCs recapitulate key pathological features of patients with SVAS and may provide a promising strategy to study disease mechanisms and to develop novel therapies.
elastin; induced pluripotent stem cells; smooth muscle alpha actin filament bundle; smooth muscle cells; supravalvular aortic stenosis
While mechanical stimuli can be used to enhance the properties of engineered cartilage, a promising alternative may be to directly harness the underlying mechanotransduction pathways responsible. Our initial studies on the adenosine triphosphate (ATP)–purinergic receptor pathway demonstrated that stimulation by exogenous ATP improved tissue growth and properties but elicited matrix turnover under high doses (250 µM) potentially due to the accumulation of extracellular inorganic pyrophosphate (ePPi). Therefore, the purpose of this study was to identify the mechanism of ATP-mediated catabolism and determine a therapeutic dose to maximize the anabolic effect.
Isolated bovine articular chondrocytes were seeded in high-density, 3-dimensional culture supplemented with varying doses of ATP for 4 weeks. The effects on biosynthesis, matrix metalloproteinase 13 (MMP-13) protein activity, and PPi accumulation were determined. Separate monolayer experiments were conducted to determine the effect of ePPi on MMP-13 activity.
High doses of ATP resulted in an increase in ePPi accumulation (by 54%) and MMP-13 activity (by 39%). Monolayer experiments confirmed a link between increased ePPi accumulation and MMP-13 activity, which appeared to require calcium and was inhibited by the MEK1/2 inhibitor U0126. Cultures supplemented with 62.5 to 125 µM ATP favored an anabolic response, which represented the therapeutic dose range.
A therapeutic dose range of exogenous ATP to improve the properties of engineered cartilage has been identified, and a possible catabolic mechanism involving excess PPi was determined. Future research into PPi signal transduction and pathological crystal formation is necessary to maximize the beneficial effect of exogenous ATP on chondrocyte cultures.
cartilage tissue engineering; adenosine triphosphate (ATP); calcium pyrophosphate dihydrate (CPPD); inorganic pyrophosphate (PPi); mechanotransduction
Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C68S, V80E, E94V, A160T, V176E, and V217I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C68S) to normal (V217I), with most variants demonstrating functional alteration in binding, expression or activation (V80E, E94V, A160T, and V176E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and “in platelet” evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.
Cardiovascular disease is the foremost cause of morbidity and mortality in the western world. Atherosclerosis followed by thrombosis (atherothrombosis) is the pathological process underlying most myocardial, cerebral, and peripheral vascular events. Atherothrombosis is a complex and heterogeneous inflammatory process that involves interactions between many cell types (including vascular smooth muscle cells, endothelial cells, macrophages and platelets) and processes (including migration, proliferation, and activation). Despite a wealth of knowledge from many recent studies using knockout (KO) mouse and human genetic studies (GWAS and candidate approach) identifying genes and proteins directly involved in these processes, traditional cardiovascular risk factors (hyperlipidemia, hypertension, smoking, diabetes mellitus, sex and age) remain the most useful predictor of disease. Eicosanoids (20 carbon polyunsaturated fatty acid derivatives of arachidonic acid and other essential fatty acids) are emerging as important regulators of cardiovascular disease processes. Drugs indirectly modulating these signals, including COX-1/COX-2 inhibitors, have proven to play major roles in the atherothrombotic process. However, the complexity of their roles and regulation by opposing eicosanoid signaling, have contributed to the lack of therapies directed at the eicosanoid receptors themselves. This is likely to change, as our understanding of the structure, signaling and function of the eicosanoid receptors improves. Indeed, a major advance is emerging from the characterization of dysfunctional naturally occurring mutations of the eicosanoid receptors. In light of the proven and continuing importance of risk factors we have elected to focus on the relationship between eicosanoids and cardiovascular risk factors.
eicosanoids; atherothrombosis; prostaglandins; prostanoids; platelets; hypertension; hyperlipidemia; oxidative stress; diabetes mellitus
Myocardial hypertrophy is an adaptation to increased hemodynamic demands. An increase in heart tissue must be matched by a corresponding expansion of the coronary vasculature to maintain and adequate supply of oxygen and nutrients for the heart. The physiological mechanisms that underlie the coordination of angiogenesis and cardiomyocyte growth are unknown. We report that induction of myocardial angiogenesis promotes cardiomyocyte growth and cardiac hypertrophy through a novel NO-dependent mechanism. We used transgenic, conditional overexpression of placental growth factor (PlGF) in murine cardiac tissues to stimulate myocardial angiogenesis and increase endothelial-derived NO release. NO production, in turn, induced myocardial hypertrophy by promoting proteasomal degradation of regulator of G protein signaling type 4 (RGS4), thus relieving the repression of the Gβγ/PI3Kγ/AKT/mTORC1 pathway that stimulates cardiomyocyte growth. This hypertrophic response was prevented by concomitant transgenic expression of RGS4 in cardiomyocytes. NOS inhibitor L-NAME also significantly attenuated RGS4 degradation, and reduced activation of AKT/mTORC1 signaling and induction of myocardial hypertrophy in PlGF transgenic mice, while conditional cardiac-specific PlGF expression in eNOS knockout mice did not induce myocardial hypertrophy. These findings describe a novel NO/RGS4/Gβγ/PI3Kγ/AKT mechanism that couples cardiac vessel growth with myocyte growth and heart size.
Adiponectin is a cardioprotective adipokine derived predominantly from visceral fat. We recently demonstrated that exogenous adiponectin induces vascular smooth muscle cell (VSMC) differentiation via repression of mTORC1 and FoxO4. Here we report for the first time that VSMC express and secrete adiponectin, which acts in an autocrine and paracrine manner to regulate VSMC contractile phenotype. Adiponectin was found to be expressed in human coronary artery and mouse aortic VSMC. Importantly, siRNA knock-down of endogenous adiponectin in VSMC significantly reduced the expression of VSMC contractile proteins. Contractile protein deficiency was also observed in primary VSMC isolated from Adiponectin-/- mice. This deficiency could be rescued by culturing Adiponectin-/- VSMC in conditioned media from wild type (WT) VSMC. Moreover, the paracrine effect of VSMC-derived adiponectin was confirmed as adiponectin neutralizing antibody blocked the rescue. Overexpressed adiponectin also exerted paracrine effects on neighboring untransfected VSMC, which was also blocked by adiponectin neutralizing antibody. Interestingly, adiponectin expression was inducible by the PPARγ agonist rosiglitazone. Our data support an important role for VSMC-derived adiponectin in maintaining VSMC contractile phenotype, contributing to critical cardioprotective functions in the vascular wall.
Adiponectin; vascular smooth muscle; contractile proteins; contractile phenotype; autocrine; paracrine
The adipocyte-secreted hormone adiponectin exerts important cardioprotective and anti-diabetic effects. Little is known about its effect on vascular smooth muscle cells (VSMC), key cells in restenosis, hypertension, and atherosclerosis.
Methods and Results
Using human coronary artery VSMC, we report that recombinant adiponectin in the HMW or trimeric, but not globular forms induces VSMC differentiation through a mechanism similar to the classic feedback signaling employed by rapamycin, a drug known to effectively inhibit restenosis on drug-eluting stents (DES). Using a combination of pharmacologic agents, siRNA, and overexpression approaches, we demonstrate that adiponectin activates 5′ AMP-activated protein kinase (AMPKα2), leading to inhibition of mammalian target of rapamycin complex 1 (mTORC1) and S6K1. This in turn stabilizes IRS-1, driving Akt2 -mediated inhibition of FoxO4 and subsequent contractile protein induction. While adiponectin and rapamycin have similarly beneficial effects on VSMC phenotype in both cell and organ culture, a direct comparison of the effects of rapamycin versus adiponectin on endothelial cells (EC) revealed distinct differences: rapamycin inhibited, while adiponectin maintained, Akt phosphorylation. Importantly, Akt activity preserves endothelial function.
Adiponectin promotes VSMC differentiation and preserves EC Akt signaling, suggesting that targeting the adiponectin pathway may have advantages over rapamycin in developing new DES therapeutics.
Adiponectin; VSMC; differentiation; mTOR; rapamycin; AMPK; Akt2; FoxO4
Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 22.214.171.124), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications.
aldose reductase; oxidative stress; diabetes mellitus; atherosclerosis; thrombosis
Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure.
The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species), was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3′-N-X-P-Y-M-G-L-5′, which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3′ leader RNA of 149 nt and a 5′ trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine the subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.
rhabdovirus; GFP; TagRFP; Nicotiana benthamiana; BiFC; interactome; localization; FRAP; confocal; nuclear localization
Prostacyclin (PGI2) is a member of the prostaglandin family of bioactive lipids. Its best-characterized role is in the cardiovascular system, where it is released by vascular endothelial cells, serving as a potent vasodilator and inhibitor of platelet aggregation. In recent years, prostacyclin (PGI2) has also been shown to promote differentiation and inhibit proliferation in vascular smooth muscle cells. In addition to these well-described homeostatic roles within the cardiovascular system, prostacyclin (PGI2) also plays an important role as an inflammatory mediator. In this review, we focus on the contribution of prostacyclin (PGI2) as both a pathophysiological mediator and therapeutic agent in three major inflammatory-mediated disease processes, namely rheumatoid arthritis, where it promotes disease progression (“pro-inflammatory”), along with pulmonary vascular disease and atherosclerosis, where it inhibits disease progression (“anti-inflammatory”). The emerging role of prostacyclin (PGI2) in this context provides new opportunities for understanding the complex molecular basis for inflammatory-related diseases, and insights into the development of current and future anti-inflammatory treatments.
prostacyclin; IP receptor; inflammation; atherosclerosis; rheumatoid arthritis; pulmonary fibrosis
Exposure to the environmental toxicant arsenic, through both contaminated water and food, contributes to significant health problems worldwide. In particular, arsenic exposure is thought to function as a carcinogen for lung, skin and bladder cancer, via mechanisms that remain largely unknown. More recently, the Hedgehog (HH) signaling pathway has also been implicated in the progression and maintenance of these same cancers. Based on these similarities, we tested the hypothesis that arsenic may act in part through activating HH signaling. Here, we show that arsenic is able to activate HH signaling in a number of primary and established tissue culture cells, as well as in vivo. Arsenic activates HH signaling by decreasing the stability of the repressor form of GLI3, one of the transcription factors that ultimately regulate HH activity. We also show, using tumor samples from a cohort of bladder cancer patients, that high levels of arsenic exposure are associated with high levels of HH activity. Given the important role HH signaling plays in the maintenance and progression of a variety of tumors, including bladder cancer, these results suggest that arsenic exposure may in part promote cancer through the activation of HH signaling. Thus, we provide an important insight into the etiology of arsenic induced human carcinogenesis, which may be relevant to millions of people exposed to high levels of arsenic worldwide.
arsenic; Hedgehog; GLI; bladder cancer; toxicant
The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.
human vascular smooth muscle cells; iloprost; prostacyclin receptor; Akt-1; differentiation; COX-2; signaling
Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 (COX-2) inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major endproduct of COX-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in three separate Caucasian cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease, but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low risk cohort (n=2263). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared to age and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent upon the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the COX-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute in part to the underlying adverse cardiovascular outcomes observed with COX-2 inhibition.
prostacyclin; eicosanoid; cyclooxygenase-2; G-protein coupled receptor; mutation
Mammalian target of rapamycin (mTOR) activity is regulated by assembly of two functionally distinct complexes, mTORC1 and mTORC2. In syndecan-4 (S4) null endothelial cells, mTORC2 activity is reduced, resulting in decreased Akt activation, while mTORC1 activity is increased. Levels of rictor, mLST8, and mSin-1 are unchanged in total cell lysates but decreased in the rafts of S4−/− endothelial cells, as is the level of PKCα. Expression of myristoylated-PKCα in S4−/− cells restores rictor, mLST8, and mSin-1 presence in the rafts and rescues Akt phosphorylation. PKCα knockdown mimics the effect of S4 deletion on mTORC2 localization and Akt activation. Reduced mTORC2 activity in S4−/− endothelial cells results in decreased FOXO1/3a and eNOS phosphorylation, decreased endothelial cell size and increased arterial blood pressure in S4−/− mice. Thus, S4-dependent targeting of PKCα to the plasma membrane is required for recruitment of mTORC2 components to the rafts and Akt activation.
To identify compounding practices of independent community pharmacy practitioners in order to make recommendations for the development of curricular objectives for doctor of pharmacy (PharmD) programs.
Independent community practitioners were asked about compounding regarding their motivations, common activities, educational exposures, and recommendations for PharmD education.
Most respondents (69%) accepted compounding as a component of pharmaceutical care and compounded dermatological preparations for local effects, oral solutions, and suspensions at least once a week. Ninety-five percent were exposed to compounding in required pharmacy school courses and most (98%) who identified compounding as a professional service offered in their pharmacy sought additional postgraduate compounding education. Regardless of the extent of compounding emphasis in the practices surveyed, 84% stated that PharmD curricula should include compounding.
Pharmacy schools should define compounding curricular objectives and develop compounding abilities in a required laboratory course to prepare graduates for pharmaceutical care practice.
pharmaceutical care; compounding; independent community pharmacy; curricula
Hypertrophy; Shp2; FAK; mTOR
An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor.