David Shapiro - US grants

The broad objective is to examine the degree to which the regulation of blood pressure and heart rate changes during orthostatic stress can be modified by biofeedback and voluntary control procedures. A non-invasive tracking cuff system is used to measure systolic and diastolic blood pressure continuously while heart rate and respiration are simultaneously monitored. Healthy volunteers and patients with orthostatic hypotension are studied in sitting and standing conditions and while going from a sitting to a standing position. Blood pressure and/or heart rate feedback is presented to subjects by means of a computer display indicating beat-to-beat changes which may be compared with baseline or previous performance. The specific aims are to examine in healthy subjects the degree to which postural cardiovascular changes can be controlled "voluntarily", whether feedback is more effective than simple instructions only, whether different types of feedback procedures have different effects, how feedback results compare with the effects of other mental and physical strategies, whether control can be improved with repeated practice, and how orthostatic regulation varies according to age. Diabetic patients will be studied to characterize their degree of neuropathy using various tests of autonomic function, e.g. Valsalva, paced respiration, handgrip. A detailed examination will be made of the patients' response to orthostatic stress by itself and in combination with concurrent mental arithmetic and with concurrent handgrip. Out of this sample, patients will be selected with the most severe orthostatic hypotension to participate in a controlled clinical outcome study to determine whether the degree of hypotension can be modified by a biofeedback training method. Finally, several methodological issues will be investigated: comparison of different methods of assessing and quantifying arterial baroreceptor sensitivity, refinements in the blood pressure tracking system.

The broad objective of this research is to gain basic knowledge about the processes involved in the discrimination (or detection) of changes in one's own blood pressure. Male and female subjects, including normotensives and mild hypertensives, are studied under different experimental conditions. The basic questions of the research are: (1) To what extent can normal and hypertensive subjects detect variations in their own blood pressure? (2) To what extent is this ability improved by discrimination training (knowledge of results)? (3) To what extent is the discrimination performance of subjects specific to the function for which discrimination training is provided? (4) How does discrimination performance change as a function of stress-induced elevations in pressure? (5) How does discrimination performance relate to voluntary control of blood pressure? (6) How does discrimination performance relate to other selected physiological and psychological factors? The experiments are conducted under computer control. Continuous recordings are made of systolic and diastolic pressure (using a tracking-cuff system), heart rate, respiration and other physiological variables. Blood pressure discrimination is related to changes in the other physiological variables and to various subjective data obtained from post-experimental questionnaires. The research will help us understand that internal sensations and physiological processes are associated with blood pressure variability and discrimination. The research has potential application to the non-pharmacologic management of hypertension.

The broad objective of this project is to gather 24-hour ambulatory blood pressure data on healthy, normotensive men and women (ages 18-34) and to relate these data to blood pressure response to laboratory stressors. Methodological questions include: frequent vs. standard ambulatory recording rates, comparison of different rates of sampling ambulatory pressure using various level and variability indices, day-by-day and year- by-year consistency of ambulatory records, investigation of different methods of quantifying and analyzing pressure level and variability. Substantive questions include: comparison of lab and natural setting blood pressure, differences between men and women in lab and ambulatory responses, relations between family history and blood pressure according to age/sex norms and lab/ambulatory blood pressure in initial and follow-up observations. The lab stressors include; mental arithmetic, video game, cold pressor test, and postural change. Lab measures include: continuous systolic and diastolic pressure, heart rate, skin conductance, and cardiac and flow measures using impedance cardiography. This project should provide "normative" data on blood pressure level and variability relevant to the use of ambulatory monitoring in the assessment of hypertension and in the evaluation of effects of drug and/or behavioral treatments. Results of the methodological studies should also lead to improved methods of acquiring and analyzing ambulatory blood pressure. The research on lab and natural blood pressure and ther cardiovascular processes under different stress conditions and according to several risk factors should provide significant information on the regulation of blood presssure in normotensive individuals and in the development and pathogenesis of hypertension.

The major goal of this project is to determine if adding a cognitive-behavioral treatment to the drug treatment of mild hypertension will allow reductions in anti-hypertensive medication levels required to maintain blood pressure at normotensive limits (< 90 mmHg diastolic BP DBP). The cognitive-behavioral treatment is compared to a control involving all study measurements only, including self-recording of BP by patients at home and ambulatory 24-hour recording. The research involves several important methodological departures from previous work: (1) patients are thoroughly evaluated prior to the study to determine if their DBP is > 95 mmHg, unmedicated; (2) a systematic drug protocol is used to establish minimal drug requirements; (3) after the intervention phase, all patients (treatment and control.) are systematically tapered off their medication to determine minimal levels to maintain control; (4) detailed evaluations of psychosocial quality of life, health habits, and short-term memory are made periodically throughout the study; (5) long-term follow-up of all patients. To date, 10 treatment and 11 control patients have completed the intervention phase. Within three months of follow-up, 5 out of the 11 controls have returned to initial medication levels, while all-treatment patients have been able to reduce their medication requirements. In the continuation, we propose to complete the initially proposed sample to 30 treatment and 30 controls, follow-up treatment group patients for 2 years, cross over control patients to the treatment condition, compare intervention successes and failures, and provide detailed evaluations of quality of life, memory, health habits, and multiple assessments of BP (clinic, ambulatory, home) as they relate to treatment-control differences and changes over various phases of the study.

DESCRIPTION: (Applicant's Abstract) The pharmacologic basis of smoking has received a lot of attention with development of nicotine replacement for smoking cessation. Long-term abstinence achieved by nicotine replacement has been modest, suggesting that smoking involves non-nicotinic factors. The behavior of smoking is subject to conditioning processes and comes under the control of external and internal stimuli that serve as cues for smoking. The main objective of this project is to expand our knowledge of the cues associated with smoking in everyday settings. Data on these associations will be provided by smokers using a diary to monitor their location, activity, moods, and social setting during two work and two nonwork days at times they smoke and at control non-smoking occasions. In addition, subjects will wear devices that record heart rate and physical activity continuously during each day to determine how these measures are associated with smoking. In a second set of four days of recording, subjects will wear either a nicotine or a placebo patch to determine how associations between smoking and the diary and physiological measures are affected by added nicotine. We will also determine the role of smoking rate, gender, nicotine dependence, personality and other characteristics in the above relationships. The specific aims are: 1. To evaluate the stimulus control of smoking in everyday situations using ambulatory methods. 2. To determine whether stimulus control differs as a function of smoking rate, gender, and individual differences in nicotine dependence, hostility, and other characteristics. 3. To evaluate the consistency of stimulus control of smoking from day to day and as a function of social context (work, nonwork days). 4. To evaluate the effects of added nicotine administered by patch compared to a placebo on the stimulus control of smoking and the relation of these effects to gender, smoking rate, and nicotine dependence. 5. To explore the role of heart rate and physical activity level in association with smoking. 6. To explore the role of personality traits (hostility, anxiety, depression) and other characteristics in the above rdationships. Participants in the study will be 60 men and 60 women heavy and moderate smokers. This research should add to our knowledge of non-nicotinic factors in smoking and help in the design of more effective smoking cessation programs.

DESCRIPTION (provided by applicant): We propose a novel approach to identifying new probes and potential anticancer agents based on reducing expression of c-Myc and other oncogenes, MDR1 (multidrug resistance protein 1) and NF-?B by targeting the mRNA binding protein IMP-1/CRD-BP/IGF2BP1. IMP-1 is an oncofetal mRNA binding protein that binds to and stabilizes the mRNAs encoding c-Myc, K-Ras, ERK and other oncogenes, MDR1, and indirectly increases activity of the tumor enabling factor NF-?B. ?-catenin and c-Myc induce IMP-1 and it is a key regulatory target of let-7 microRNA. Reducing IMP-1 levels by RNAi knockdown, or by expression of let-7 miRNA, reduces c- Myc levels, strongly inhibits cell proliferation and reverses resistance to anticancer drugs. Kaplan-Meier plots show that expression of IMP-1 is associated with a poor prognosis and reduced survival in lung, ovarian and colon cancer. Small molecule inhibitors of IMP-1 have not been reported. In preliminary studies, we identified an IMP-1 binding and mRNA stabilizing site in MDR1 mRNA, established a (FAMA) fluorescence anisotropy/polarization microplate assay for analyzing binding of IMP-1 to its c-Myc and MDR1 mRNA targets, developed a robust (Z' factor=0.64) FAMA-based high throughput screen for inhibitors of binding of IMP-1 to a c-Myc mRNA binding site and carried out a successful pilot screen. To filter the primary hits, we established verification assays based on purified protein, luciferase reporters and cell-proliferation. The Specific Aims are: Aim 1. To identify small molecules with high potency and specificity that inhibit binding of IMP-1 to its c-Myc mRNA binding site. We will implement a FAMA HTS screen using ~180,000 small molecules and identify compounds that inhibit binding of purified IMP-1 to a fluorescein-labeled (fl) c-Myc mRNA binding site. To reduce the number of false positives, our two-step assay first uses an internal counterscreen to test whether each compound reduces the signal from the fl-Myc probe alone and then tests whether the compound inhibits binding of IMP-1 to the fl-Myc probe. (i) Primary hits will be simultaneously verified using fl-Myc and tested for ability to inhibit bindingof IMP-1 to the fl-MDR1 mRNA binding site (ii) To test for specificity, we will evaluate hits for inhibition of binding of progesterone receptor (PR) to its fl-DNA response element (fl-PRE). (iii) Potency and efficacy of hits will be evaluated in dose-response studies. Aim 2. To identify lead IMP-1 inhibitors which reduce the growth of cancer cells. Initial cell-based assays are luciferase-based assays for small molecules that inhibit expression of NF-?B-luciferase and our luciferase- MDR1 mRNA chimera and for inhibition of proliferation of IMP-1 positive IGROV-1 and ES-2 ovarian cancer cells with little or no effect on IMP-1 negative PC-3 cells. To approach inhibitor sites of action, lead compounds will be tested for down-regulation of IMP-1 protein and c-Myc and MDR1 mRNA and protein. Microarray studies using IMP-1 negative cells will assess possible off-target effects of the lead inhibitor. Lead structures will be confirmed, leads resynthesized and structural studies of inhibitor bound to IMP-1 will be initiated.

Estrogens, acting via estrogen receptor ? (ER?), were known to regulate gene expression and to activate signal transduction pathways. We identified a conserved extranuclear pathway by which 17?-estradiol (E2), acting through ER?, rapidly activates phosphoplipase C ? (PLC?) leading to production of inositol triphosphate (IP3). The IP3 binds to and opens endoplasmic reticulum (EnR) IP3 receptors (IP3R) leading to extremely rapid (<1 min.) efflux of calcium (Ca2+) from the lumen of the EnR into the cell body. Elevated intracellular Ca2+ primes cells for subsequent actions of E2-ER?; depletion of EnR Ca2+ activates the unfolded protein response (UPR), inducing the important chaperone BiP/GRP78 (glucose regulated protein 78 kDa). Activation of this pathway is required for E2-ER?-regulated gene expression, induction of cell proliferation and protects cells against stress. We target this pathway with our medically promising ER? biomodulator, BHPI, which uses the same pathway as E2, but induces toxic hyperactivation of the UPR. Our hypothesis is that the products of activation of this newly unveiled pathway, elevated intracellular calcium (Aim 1), and at later times, BiP chaperone (Aim 2), link to and regulate subsequent E2-ER?-regulated gene expression and stabilize ER?, influencing drug resistance and genomic actions of ER?. Our goals are to identify the mechanism(s) by which these products couple to, and control, gene expression (Aim 1), ER? stability and response to drugs (Aim 2), and to identify the sensors and signals that allow E2-ER? to rapidly initiate the pathway (Aim 3). Aim 1. Identify the mechanism(s) by which the product of E2-ER? activation of the pathway couples to and controls E2-ER?-regulated gene expression. Test the data-driven hypothesis that Ca2+ produced by pathway activation acts through the Ca2+ sensor calmodulin (CaM) to regulate nuclear E2-ER?:CaM interaction, E2-ER? dimerization and nuclear localization and thereby controls E2-ER?-regulated gene expression. Aim 2. Background: In CRISPR/Cas9 generated cell lines expressing constitutively active ER? mutants, the UPR is activated and ER? is partially resistant to antagonists. Identify the mechanism by which UPR activation contributes to drug resistance. Test the hypothesis that drug resistance in these cells arises in part because ER?, together with progesterone-PR, synergistically activate the UPR, inducing BiP chaperone, which stabilizes ER?, thereby contributing to drug resistant gene expression. Aim 3. Identify components of the multiprotein complex by which E2-ER? initiates the pathway. Using an unbiased CRISPR/Cas9 lethality screen, followed by verification and analysis of multiprotein complexes, we will identify the activating kinase(s), scaffolding proteins, other components of the complex(es), genes that impact the pathway and probe ER? interactions in the complex. These studies will establish the initial events that occur when estrogen contacts a cell and identify new mechanisms coupling steroid receptor regulated transcription to extranuclear signals.