Reactive oxygen species (ROS) have been shown to function as important signaling molecules in the cardiovascular system. Vascular smooth muscle cells (VSMCs) contain several sources of ROS, among which the NADPH oxidases are predominant. In VSMCs, ROS mediate many pathophysiological processes, such as growth, migration, apoptosis and secretion of inflammatory cytokines, as well as physiological processes, such as differentiation, by direct and indirect effects at multiple signaling levels. Therefore, it becomes critical to understand the different roles ROS play in the physiology and pathophysiology of VSMCs.
Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-β (TGF-β) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle á-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-β-induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.
In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.
Background: Toxoplasmosis is becoming a global health hazard as it infects 30-50% of the world human population. Clinically, the life-long presence of the parasite in tissues of a majority of infected individuals is usually considered asymptomatic. However, a number of studies show that this 'asymptomatic infection' may also lead to development of other human pathologies. Aims of the Study: The purpose of the study was to collect available geoepidemiological data on seroprevalence of toxoplasmosis and search for its relationship with mortality and disability rates in different countries. Methods and Findings: Prevalence data published between 1995-2008 for women in child-bearing age were collected for 88 countries (29 European). The association between prevalence of toxoplasmosis and specific disease burden estimated with age-standardized Disability Adjusted Life Year (DALY) or with mortality, was calculated using General Linear Method with Gross Domestic Product per capita (GDP), geolatitude and humidity as covariates, and also using nonparametric partial Kendall correlation test with GDP as a covariate. The prevalence of toxoplasmosis correlated with specific disease burden in particular countries explaining 23% of variability in disease burden in Europe. The analyses revealed that for example, DALY of 23 of 128 analyzed diseases and disease categories on the WHO list showed correlations (18 positive, 5 negative) with prevalence of toxoplasmosis and another 12 diseases showed positive trends (p < 0.1). For several obtained significant correlations between the seroprevalence of toxoplasmosis and specific diseases/clinical entities, possible pathophysiological, biochemical and molecular explanations are presented. Conclusions: The seroprevalence of toxoplasmosis correlated with various disease burden. Statistical associations does not necessarily mean causality. The precautionary principle suggests however that possible role of toxoplasmosis as a triggering factor responsible for development of several clinical entities deserves much more attention and financial support both in everyday medical practice and future clinical research.
This Critical Synthesis Package contains (1) a Critical Analysis of the psychometric properties and application to health sciences education for the Matched Pair Instrument (MPI) in Doctor-Patient Communication Skills; (2) a copy of the Matched Pair Instruments (MPI) in Doctor-Patient Communication Skills developed by Jocelyn Lockyer, PhD.
The Matched Pair Instrument in Doctor-Patient Communication Skills (MPI) is a questionnaire tool that is administered simultaneously to both the doctor and the patient following a single clinical encounter in the outpatient setting. The instrument measures the communication skills of the physician simultaneously from the perspective of both the patient and the physician. It was shown to have construct validity for use with practicing physicians; however, its validity is sensitive to both the medical context and social context in which it is used. The MPI has acceptable reliability when a large number of responses are available.
The Matched Pair Instrument for Doctor-patient Communication Skills was not designed as an educational tool and it does not have a scoring guide or performance standards that would be necessary in order to use it for educational assessment. The MPI has not been analyzed as an educational tool and it has several known limitations to its validity and reliability. The use of the MPI for instruction or educational assessment is not supported by evidence and therefore is not recommended.
The Matched Pair Instrument for Doctor-patient Communication Skills has been shown to be a reliable measure of a doctor’s communication skills, when a large number of responses are available. The validity of the instrument has been shown to be dependent on both the medical context (e.g. outpatient setting) and social context (e.g. adult, English speaking patients) in which it is used. Therefore, use of the MRI in other settings would require independent validation and a reliability analysis.
The MPI has not been analyzed as an educational tool and it has several known limitations to its validity and reliability. Use of the MPI for instruction or educational assessment is not supported by evidence and therefore is not recommended.
This resource is a guide to developing learner outcomes for a healthcare education program. The Instructor Guide describes the process of developing program outcomes; it includes three exhibits to supplement the process described in the guide. The documents showing student physician activities (SPAs), which are the outcomes developed using this process, are two versions of the same list: (1) a parsimonious list of the SPAs with reference to a national framework and (2) an explanatory list of SPAs with annotations and cross-references to other SPAs.
This resource contains two parts: 1) a Critical Analysis of the psychometric properties and application to healthcare education of the Ambati Questionnaire on Knowledge and Attitudes toward HIV/AIDS (AQ), and 2) a copy of the AQ instrument developed by Balamurali Ambati, MD, PhD.
The Ambati Questionnaire on Knowledge and Attitudes toward HIV/AIDS (AQ) is a self-report survey to measure knowledge and attitudes about AIDS patients and issues. The AQ is consists of 48 selected response items: 6 items measuring knowledge of the signs and symptoms of the disease, 12 knowledge items about transmission of HIV, and 30 items about attitudes towards HIV/AIDS patients. All items use a “yes,” “no,” or “I don’t know” response scale with “I don’t know” responses excluded from scoring. There is no scoring guide or technical manual available. Little data on internal or test-retest reliability has been published and there has been no evidence produced to establish construct or external validity of the instrument. The norming sample was heterogeneous, so applications of the instrument to healthcare education are limited. Subsequent uses of the AQ have not significantly augmented the data or psychometric support for the instrument. The AQ needs extensive work to establish validity evidence and it should be normed to a well-defined population before recommending its use in healthcare education.
The Ambati Questionnaire on Knowledge and Attitudes toward HIV/AIDS (AQ) was created based on the developers’ judgment of items that would measure the intended constructs. In the only manuscript available in MedLine journals written by the developers (Ambati B, Ambati J, Rao A. Dynamics of knowledge and attitudes about AIDS among the educated in southern India. AIDS Care. 1997 June;9(3): 319-330.), there is no reference to prior instruments or frameworks used in development. The AQ is simple to understand, administer, and score but the evidence for validity of the AQ is circumstantial. Neither the developers nor subsequent users established grounds for either construct validity or external validity of the instrument. Reporting of reliability is sketchy and the few calculated alpha coefficients are low. The “hostility index” (1) is unsupported and should not be used. The lack of technical manual or a scoring guide severely inhibits further development of the instrument for use in scholarly inquiry. Prior to using the AQ, all items used should be reviewed for validity, particularly in view of the changing social context for knowledge about of HIV/AIDS and perceptions of HIV/AIDS patients. The AQ should to be normed with a well-defined population which will allow for meaningful comparisons across time and location.
Angiotensin II receptor blockade has been shown to inhibit atherosclerosis in several different animal models. We sought to determine if this effect was the result of blood pressure reduction per se or a result of the anti-inflammatory effects of receptor blockade. ApoE-deficient mice were fed a high fat diet and treated with either an angiotensin II receptor antagonist, candesartan (0.5 mg/kg/day, SC) or a calcium channel blocker, amlodipine (7.5 mg/kg/day, mixed with food). Atherosclerotic lesion area, aortic inflammatory gene expression as well as aortic H2O2 and superoxide production were assayed. We found that candesartan but not amlodipine treatment dramatically attenuated the development of atherosclerosis despite a similar reduction in blood pressure. Similarly, candesartan treatment inhibited aortic expression of inflammatory genes and production of reactive oxygen species, effects not seen with amlodipine. These data demonstrate that angiotensin II receptor blockade inhibits atherosclerosis by reducing vascular oxidative stress and inflammatory gene production independent of blood pressure reduction.
Objective-On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. Approach and Results-Because homozygous Poldip2 deletion is lethal, Poldip2 mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2 aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2 tissue. Isolated aortas from Poldip2 mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2 mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2 mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. Conclusions-Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.