Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non-Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G-protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non-lipid raft domains of the plasma membrane, β-Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non-lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically-encoded FRET-based biosensors targeted to lipid raft and non-lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non-lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non-lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A-kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains

Targeting of ALK2, a Receptor for Bone Morphogenetic Proteins, Using the Cre/lox System to Enhance Osseous Regeneration by Adipose‐Derived Stem Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135224/1/sct320143111375.pd

Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6?8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (?CT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02?mm3, P?Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140207/1/scd.2015.0135.pd

Comparative efficacy of topical tetraVisc versus lidocaine gel in cataract surgery

<p>Abstract</p> <p>Background</p> <p>To compare the clinical efficacy of lidocaine 2% with tetracaine 0.5% for cataract surgery.</p> <p>Methods</p> <p>In a randomized, multi-surgeon, controlled clinical trial,122 consecutive cataract cases eligible for topical anesthesia, were randomly assigned to receive lidocaine 2% gel (1 ml) or tetracaine solution 0.5% (TetraVisc, 0.5 ml) before clear corneal phacoemulsification. Main outcome measure was visual analog scale (0 to 10), which was used to measure intra-operative pain. Secondary outcome measures included patients' discomfort due to tissue manipulation and surgeon graded patients' cooperation. Duration of surgery and intra-operative complications were also recorded.</p> <p>Results</p> <p>The mean age in TetraVisc (TV) group was 70.4 years and in the lidocaine gel group (LG) it was 70.6 years (p = 0.89). Patient reported mean intra-operative pain scores by visual analog scale were 0.70 ± 0.31 in TV group and 1.8 ± 0.4 in LG group (<it>P </it>< 0.001). Mean patient cooperation was also marginally better in the TV group (8.3 ± 0.3) compared to LG group (8.4 ± 0.6) (P = 0.25). 96% of patients in TV group showed intra-operative corneal clarity compared to 91% in LG group. TV group had less (1 out of 61 patients, 1.6%) intra-operative complications than LG group (3 out of 61 patients, 4.8%). No anesthesia related complications were noted in either group</p> <p>Conclusion</p> <p>Topical TetraVisc solution was superior to lidocaine 2% gel for pain control in patients undergoing clear corneal phacoemulsification. Lidocaine 2% gel is similar to TetraVisc in patient comfort and surgeon satisfaction.</p> <p>Trial Registration</p> <p><b>Clinical trials number</b>: ISRCTN78374774</p

Scleraxis‐Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136325/1/stem2515_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136325/2/stem2515.pd

Coordinating Tissue Regeneration Through Transforming Growth Factorâ β Activated Kinase 1 Inactivation and Reactivation

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factorâ β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment (â drug onâ ), the impact of drug withdrawal (â drug offâ ) implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dualâ inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment (â drug onâ ) and subsequent withdrawal (â drug offâ ) through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the â drug onâ (Creâ mediated inactivation) and â drug offâ (Flpâ mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766â 778Manipulating transforming growth factor βâ activated kinase 1 for cell and scaffold free tissue regeneration using a dualâ inducible Combinational Sequential Inversion Engineering mouse model.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149573/1/stem2991_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149573/2/stem2991.pd

Caveolin contributes to the modulation of basal and β-adrenoceptor stimulated function of the adult rat ventricular myocyte by simvastatin: A novel pleiotropic effect

The number of people taking statins is increasing across the globe, highlighting the Importance of fully understanding statins effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system (pleiotropic effects). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 μM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2]¡) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to β2-, but not β1-, adrenoceptor stimulation. Under conditions of β2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16and troponin I at Ser23/24was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive Inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered ß-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950-2019 : a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2·72 (95% uncertainty interval [UI] 2·66–2·79) in 2000 to 2·31 (2·17–2·46) in 2019. Global annual livebirths increased from 134·5 million (131·5–137·8) in 2000 to a peak of 139·6 million (133·0–146·9) in 2016. Global livebirths then declined to 135·3 million (127·2–144·1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2·1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27·1% (95% UI 26·4–27·8) of global livebirths. Global life expectancy at birth increased from 67·2 years (95% UI 66·8–67·6) in 2000 to 73·5 years (72·8–74·3) in 2019. The total number of deaths increased from 50·7 million (49·5–51·9) in 2000 to 56·5 million (53·7–59·2) in 2019. Under-5 deaths declined from 9·6 million (9·1–10·3) in 2000 to 5·0 million (4·3–6·0) in 2019. Global population increased by 25·7%, from 6·2 billion (6·0–6·3) in 2000 to 7·7 billion (7·5–8·0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58·6 years (56·1–60·8) in 2000 to 63·5 years (60·8–66·1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019