Why Language Matters in Digital Endpoint Development: Harmonized Terminology as a Key Prerequisite for Evidence Generation.

Background: Developments in the field of digital measures and digitally derived endpoints demand greater attention on globally aligned approaches to enhance digital measure acceptance by regulatory authorities and health technology assessment (HTA) bodies for decision-making. In order to maximize the value of digital measures in global drug development programs and to ensure study teams and regulators are referring to the same items, greater alignment of concepts, definitions, and terminology is required. This is a fast-moving complex field; every day brings new technologies, algorithms, and possibilities. A common language is particularly important when working in multifunctional teams to ensure that there is a clear understanding of what is meant and understood. Summary: In the paper, the EFPIA digital endpoint joint subgroup reviews the challenges facing teams working to advance digital endpoints, where different terms are used to describe the same things, where common terms such as "monitoring" have significantly different meaning for different regulatory agencies, where the preface "e" to denote electronic is still used in some contexts, but the term "digital" is used in other, and where there is significant confusion as to what is understood by "raw" when it comes to data derived from digital health technologies. Key Message: The EFPIA subgroup is calling for an aligned lexicon. Alignment provides a more predictable path for development, validation, and use of the tools and measures used to collect digital endpoints supporting standardization and consistency in this new field of research, with the goal of increasing regulatory and payer harmonization and acceptance.

A patient-centred conceptual model of nocturnal scratch and its impact in atopic dermatitis: A mixed-methods study supporting the development of novel digital measurements.

Background: Emerging digital measures and clinical outcome assessments (COAs) leveraging digital health technologies (DHTs) could address the need for objective, quantitative measures of symptoms of atopic dermatitis (AD), such as nocturnal scratching. Development of such measures needs to be supported by evidence reflecting meaningfulness to patients. Objectives: To assess nocturnal scratching as a concept of interest associated with meaningful aspects of health of patients with AD (adults and children); and to explore patient-centred considerations for novel COAs measuring nocturnal scratch using DHTs. Methods: Phase 1 evaluated disease impacts on everyday life and the lived experience with nocturnal scratching through qualitative interviews of AD patients and caregivers. Phase 2 deployed a quantitative survey to a sample of AD patients as well as caregivers. Results: Four cohorts with various AD severity levels participated in Phase 1: (1) adults with AD (n = 15), (2) their caregivers/spouses/partners (n = 6), (3) children with AD (n = 14), and (4) their adult caregivers (n = 14). Findings were used to develop a conceptual model for nocturnal scratching as a potential concept of interest. The Phase 2 survey was completed by 1349 of 27640 invited adults with AD and caregivers of children with AD. The most burdensome aspects of AD reported were itchy skin and scratching. Overall, ∼65% of participants reported nocturnal scratching ≥1 day/week, resulting in ∼1-1.4 h of sleep lost per night. In all, 85%-91% of respondents considered it at least somewhat valuable that a treatment reduces night-time scratching. About 50% reported willingness to use technology to this end and ∼25% were unsure. Conclusion: Our results represented by the conceptual model confirm that nocturnal scratch is a concept of interest related to meaningful aspects of health for patients with AD and therefore is worth being captured as a distinct outcome for clinical and research purposes. DHTs are suitable tools presenting an important measurement opportunity to assess and evaluate occurrence, frequency, and other parameters of nocturnal scratching as a disease biomarker or COA of treatment efficacy.

Recommendations for Defining and Reporting Adherence Measured by Biometric Monitoring Technologies: Systematic Review.

BACKGROUND: Suboptimal adherence to data collection procedures or a study intervention is often the cause of a failed clinical trial. Data from connected sensors, including wearables, referred to here as biometric monitoring technologies (BioMeTs), are capable of capturing adherence to both digital therapeutics and digital data collection procedures, thereby providing the opportunity to identify the determinants of adherence and thereafter, methods to maximize adherence. OBJECTIVE: We aim to describe the methods and definitions by which adherence has been captured and reported using BioMeTs in recent years. Identifying key gaps allowed us to make recommendations regarding minimum reporting requirements and consistency of definitions for BioMeT-based adherence data. METHODS: We conducted a systematic review of studies published between 2014 and 2019, which deployed a BioMeT outside the clinical or laboratory setting for which a quantitative, nonsurrogate, sensor-based measurement of adherence was reported. After systematically screening the manuscripts for eligibility, we extracted details regarding study design, participants, the BioMeT or BioMeTs used, and the definition and units of adherence. The primary definitions of adherence were categorized as a continuous variable based on duration (highest resolution), a continuous variable based on the number of measurements completed, or a categorical variable (lowest resolution). RESULTS: Our PubMed search terms identified 940 manuscripts; 100 (10.6%) met our eligibility criteria and contained descriptions of 110 BioMeTs. During literature screening, we found that 30% (53/177) of the studies that used a BioMeT outside of the clinical or laboratory setting failed to report a sensor-based, nonsurrogate, quantitative measurement of adherence. We identified 37 unique definitions of adherence reported for the 110 BioMeTs and observed that uniformity of adherence definitions was associated with the resolution of the data reported. When adherence was reported as a continuous time-based variable, the same definition of adherence was adopted for 92% (46/50) of the tools. However, when adherence data were simplified to a categorical variable, we observed 25 unique definitions of adherence reported for 37 tools. CONCLUSIONS: We recommend that quantitative, nonsurrogate, sensor-based adherence data be reported for all BioMeTs when feasible; a clear description of the sensor or sensors used to capture adherence data, the algorithm or algorithms that convert sample-level measurements to a metric of adherence, and the analytic validation data demonstrating that BioMeT-generated adherence is an accurate and reliable measurement of actual use be provided when available; and primary adherence data be reported as a continuous variable followed by categorical definitions if needed, and that the categories adopted are supported by clinical validation data and/or consistent with previous reports.

Development of digital measures for nighttime scratch and sleep using wrist-worn wearable devices.

Patients with atopic dermatitis experience increased nocturnal pruritus which leads to scratching and sleep disturbances that significantly contribute to poor quality of life. Objective measurements of nighttime scratching and sleep quantity can help assess the efficacy of an intervention. Wearable sensors can provide novel, objective measures of nighttime scratching and sleep; however, many current approaches were not designed for passive, unsupervised monitoring during daily life. In this work, we present the development and analytical validation of a method that sequentially processes epochs of sample-level accelerometer data from a wrist-worn device to provide continuous digital measures of nighttime scratching and sleep quantity. This approach uses heuristic and machine learning algorithms in a hierarchical paradigm by first determining when the patient intends to sleep, then detecting sleep-wake states along with scratching episodes, and lastly deriving objective measures of both sleep and scratch. Leveraging reference data collected in a sleep laboratory (NCT ID: NCT03490877), results show that sensor-derived measures of total sleep opportunity (TSO; time when patient intends to sleep) and total sleep time (TST) correlate well with reference polysomnography data (TSO: r = 0.72, p < 0.001; TST: r = 0.76, p < 0.001; N = 32). Log transformed sensor derived measures of total scratching duration achieve strong agreement with reference annotated video recordings (r = 0.82, p < 0.001; N = 25). These results support the use of wearable sensors for objective, continuous measurement of nighttime scratching and sleep during daily life.

Sleep Disturbances and Atopic Dermatitis: Relationships, Methods for Assessment, and Therapies.

Atopic dermatitis is one of the most common chronic inflammatory skin conditions and is associated with sleep disturbances in 47% to 80% of children and 33% to 90% of adults. Herein, we review the literature on sleep disturbances experienced by patients with atopic dermatitis, as well as the mechanisms that may underlie this. We present subjective and objective methods for measuring sleep quantity and quality and discuss strategies for management. Unfortunately, the literature on this topic remains sparse, with most studies evaluating sleep as a secondary outcome using subjective measures. The development of portable, at-home methods for more objective measures offers new opportunities to better evaluate sleep disturbances in atopic dermatitis research studies and in clinical practice.

Fit-for-Purpose Biometric Monitoring Technologies: Leveraging the Laboratory Biomarker Experience.

Biometric monitoring technologies (BioMeTs) are becoming increasingly common to aid data collection in clinical trials and practice. The state of BioMeTs, and associated digitally measured biomarkers, is highly reminiscent of the field of laboratory biomarkers 2 decades ago. In this review, we have summarized and leveraged historical perspectives, and lessons learned from laboratory biomarkers as they apply to BioMeTs. Both categories share common features, including goals and roles in biomedical research, definitions, and many elements of the biomarker qualification framework. They can also be classified based on the underlying technology, each with distinct features and performance characteristics, which require bench and human experimentation testing phases. In contrast to laboratory biomarkers, digitally measured biomarkers require prospective data collection for purposes of analytical validation in human subjects, lack well-established and widely accepted performance characteristics, require human factor testing, and, for many applications, access to raw (sample-level) data. Novel methods to handle large volumes of data, as well as security and data rights requirements add to the complexity of this emerging field. Our review highlights the need for a common framework with appropriate vocabulary and standardized approaches to evaluate digitally measured biomarkers, including defining performance characteristics and acceptance criteria. Additionally, the need for human factor testing drives early patient engagement during technology development. Finally, use of BioMeTs requires a relatively high degree of technology literacy among both study participants and healthcare professionals. Transparency of data generation and the need for novel analytical and statistical tools creates opportunities for precompetitive collaborations.

Preclinical to Clinical Translation of Hemodynamic Effects in Cardiovascular Safety Pharmacology Studies.

Cardiovascular (CV) safety-related attrition is an important contributor to the loss of promising drug candidates during development. CV safety pharmacology studies are conducted to identify these safety effects. Understanding translation of CV endpoints (specifically, heart rate [HR], and blood pressure [BP]) across preclinical animal models and to the clinic is critical in developing a robust CV derisking strategy. To this end, we investigated translation of HR and BP endpoints using data from 83 compounds that were tested in telemetry studies in rat and large animal (LA; dog or monkey) and 79 compounds that were tested in LA telemetry studies and human phase I clinical trials. Sensitivity, specificity as well as predictive values were calculated for rat to LA model comparison and for LA to human studies comparison. The rat CV model showed good concordance (sensitivity = 84% and specificity = 71%) for LA BP and HR changes. Similarly, LA CV measures of HR and BP showed good concordance (sensitivity = 78% and specificity = 79%) to clinical changes. The CV effects generally occurred within 0.3-3× free plasma concentration across species. Directionality of BP and HR change was conserved between LA to humans. However, for rat to LA comparisons the directionality of change was opposite for 23%-26% compounds. In conclusion, these data establish the translation of HR and BP from preclinical to clinical studies and emphasize the importance of preclinical animal models in the examination of CV safety of drugs.

Physiological approaches to assess diminished sympathetic activity in the conscious rat.

The purpose of this study was to evaluate functional measures of diminished sympathetic activity after postganglionic neuronal loss in the conscious rat. To produce variable degrees of sympathetic postganglionic neuronal loss, adult rats were treated daily with toxic doses of guanethidine (100mg/kg) for either 5days or 11days, followed by a recovery period of at least 18days. Heart rate, blood pressure, cardiac baroreflex responsiveness, urinalysis (for catecholamine metabolite, 3-methoxy-4-hydroxyphenylethylenglycol; MHPG), and pupillometry were performed during the recovery period. At the end of the recovery period stereology of superior cervical ganglia (SCG) was performed to determine the degree of neuronal loss. Total number of SCG neurons was correlated to physiological outcomes using regression analysis. Whereas guanethidine treatment for 11days caused significant reduction in the number of neurons (15,646±1460 vs. 31,958±1588), guanethidine treatment for 5days caused variable levels of neuronal depletion (26,009±3518). Regression analysis showed that only changes in urinary MHPG levels and systolic blood pressure significantly correlated with reduction of SCG neurons (r2=0.45 and 0.19, both p<0.05). Although cardiac baroreflex-induced reflex tachycardia (345.7±19.6 vs. 449.7±20.3) and pupil/iris ratio (0.50±0.03% vs. 0.61±0.02%) were significantly attenuated in the 11-day guanethidine treated rats there was no significant relationship between these measurements and the number of remaining SCG neurons after treatment (p>0.05). These data suggest that basal systolic blood pressure and urinary MHPG levels predict drug-induced depletion of sympathetic activity in vivo.

Chronic exposure to low doses of estradiol-17ß increases blood pressure in young female rats: A possible role for central Endothelin-1.

Previously, we demonstrated that chronic exposure to low levels of estradiol-17ß (E2) increases mean arterial pressure (MAP) in young female Sprague-Dawley (SD) rats, however, the underlying mechanisms are unclear. Since endothelin-1 (ET-1) is implicated in blood pressure (BP) regulation, we hypothesized that E2's effects on MAP are mediated through central ET-1. To test this, young female SD rats were either sham implanted or implanted s.c. with slow-release E2 pellets (20 ng/day for 90 days). BP was monitored by telemetry. After 75 days of E2 exposure, ETA antagonist or vehicle was administered i.c.v. After 90 days of E2 exposure, rats were sacrificed, and the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) were microdissected for gene expression and protein analysis of ET-1 and its receptors. E2 exposure increased MAP after pellet implantation. Gene expression of ET-1 and ETA but not ETB receptors were upregulated in the PVN and RVLM of E2 treated animals. Further, the protein levels of ETA receptor were also increased in the PVN of E2 treated animals. However, i.c.v. infusion of the ETA antagonist did not completely block the increase in blood pressure. Our results suggest that increases in central ET-1 activity could possibly play a role in chronic E2-induced increase in BP but further studies are needed to completely understand the contribution of ET-1 in this phenomenon.

Discovery of the Potent and Selective M1 PAM-Agonist N-[(3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4-yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of Efficacy and Cholinergic Side Effects.

It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer's disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

Toxicity of hydroxyurea in rats and dogs.

The toxicity of hydroxyurea, a treatment for specific neoplasms, sickle-cell disease, polycythemia, and thrombocytosis that kills cells in mitosis, was assessed in repeat-dose, oral gavage studies in rats and dogs and a cardiovascular study in telemetered dogs. Hydroxyurea produced hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity with steep dose response curves. In rats dosed for 10 days, 50 mg/kg/day was tolerated; 500 mg/kg/day produced decreased body weight gain; decreased circulating leukocytes, erythrocytes, and platelets; decreased cellularity of thymus, lymph nodes, and bone marrow; and epithelial degeneration and/or dysplasia of the stomach and small intestine; 1,500 mg/kg/day resulted in deaths on day 5. In dogs, a single dose at ≥ 250 mg/kg caused prostration leading to unscheduled euthanasia. Dogs administered 50 mg/kg/day for 1 month had decreased circulating leukocytes, erythrocytes, and platelets; increased bone marrow cellularity with decreased maturing granulocytes; increased creatinine kinase activity; and increased iron pigment in bone marrow and hepatic sinusoidal cells. In telemetered dogs, doses ≥ 15 mg/kg decreased systolic blood pressure (BP); 50 mg/kg increased diastolic BP, heart rate, and change in blood pressure over time (+dP/dt), and decreased QT and PR intervals and maximum left ventricular systolic and end diastolic pressures with measures returning to control levels within 24 hr.

Cyclooxygenase-1 inhibition attenuates angiotensin II-salt hypertension and neurogenic pressor activity in the rat.

Cyclooxygenase (COX)-derived prostanoids contribute to angiotensin II (ANG II) hypertension (HTN). However, the specific mechanisms by which prostanoids act are unclear. ANG II-induced HTN is caused partly by increased sympathetic nervous system activity, especially in a setting of high dietary salt intake. This study tested the hypothesis that COX-derived prostanoids cause ANG II-salt sympathoexcitation and HTN. Experiments were conducted in conscious rats. Infusion of ANG II (150 ng·kg(-1)·min(-1) sc) caused a marked HTN in rats on 2% salt diet, but a much smaller increase in blood pressure in rats on 0.4% salt diet. The nonselective COX inhibitor ketoprofen (2 mg/kg sc) given throughout the ANG-II infusion period attenuated HTN development in rats on 2% NaCl diet, but not in rats on 0.4% NaCl diet. The acute depressor response to ganglion blockade was used to assess neurogenic pressor activity in rats on 2% NaCl diet. Ketoprofen-treated rats showed a smaller fall in arterial pressure in response to ganglion blockade during ANG-II infusion than did nontreated controls. In additional experiments, ketoprofen-treated rats exhibited smaller increases in plasma norepinephrine levels and whole body norepinephrine spillover than we previously reported in ANG II-salt HTN. Finally, the effects of the selective COX-1 inhibitor SC560 (10 mg·kg(-1)·day(-1) ip) and the selective COX-2 inhibitor nimesulide (10 mg·kg(-1)·day(-1) ip) were investigated. Treatment with SC560 but not nimesulide significantly reduced blood pressure and the depressor response to ganglion blockade in ANG II-salt HTN rats. The results suggest that COX-1 products are critical for sympathoexcitation and the full development of ANG II-salt HTN in rats.

Nitric oxide synthase, ADMA, SDMA, and nitric oxide activity in the paraventricular nucleus throughout the etiology of renal wrap hypertension.

Within the paraventricular nucleus (PVN), there is a balance between the excitatory and inhibitory neurotransmitters that regulate blood pressure; in hypertension, the balance shifts to enhanced excitation. Nitric oxide (NO) is an atypical neurotransmitter that elicits inhibitory effects on cardiovascular function. We hypothesized that reduced PVN NO led to elevations in blood pressure during both the onset and sustained phases of hypertension due to decreased NO synthase (NOS) and increased asymmetrical dimethylarginine (ADMA; an endogenous NOS inhibitor) and symmetric dimethylarginine (SDMA). Elevated blood pressure, in response to PVN bilateral microinjections of a NO inhibitor, nitro-L-arginine methyl ester, was blunted in renal wrapped rats during the onset of hypertension (day 7) and sustained renal wrap hypertension (day 28) compared with sham-operated rats. Adenoviruses (Ad) encoding endothelial NOS (eNOS) or LacZ microinjected into the PVN [1 × 10(9) plaque-forming units, bilateral (200 nl/site)] reduced mean arterial pressure compared with control (Day 7, Ad LacZ wrap: 144 ± 7 mmHg and Ad eNOS wrap: 117 ± 5 mmHg, P ≤ 0.05) throughout the study (Day 28, Ad LacZ wrap: 123 ± 1 mmHg and Ad eNOS wrap: 108 ± 4 mmHg, P ≤ 0.05). Western blot analyses of PVN NOS revealed significantly lower PVN neuronal NOS during the onset of hypertension but not in sustained hypertension. Reduced SDMA was found in the PVN during the onset of hypertension; however, no change in ADMA was observed. In conclusion, functional indexes of NO activity indicated an overall downregulation of NO in renal wrap hypertension, but the mechanism by which this occurs likely differs throughout the development of hypertension.

The development of hypertension and hyperaldosteronism in a rodent model of life-long obesity.

Aldosterone has been linked to the deleterious cardiovascular effects of obesity in humans. The association of aldosterone with obesity in rodents is less well defined, particularly in models of diet-induced obesity. We hypothesized that adrenal aldosterone production and aldosterone synthase expression would be increased in rats with obesity-induced hypertension. Male Sprague Dawley rats were fed a high-fat (HF: 36% fat) or control diet from 3 wk of age, and mean arterial pressure (MAP) was measured by telemetry. MAP was increased after 4 wk of HF diet; this was 6 wk before changes in body weight. Mineralocorticoid receptor antagonism did not prevent the HF-induced increase in MAP. After 17 wk on the diets, HF rats had increased body and fat weights (abdominal and epididymal) and were insulin resistant (Homeostasis Model Assessment index: 3.53 ± 0.43 vs. 8.52 ± 1.77; control vs. HF, P < 0.05). Plasma aldosterone levels were increased in the HF rats (64.14 ± 14.96 vs. 206.25 ± 47.55 pg/ml; control vs. HF, P < 0.05). This occurred independently of plasma renin activity (4.8 ± 0.92 vs. 4.73 ± 0.66 ng/ml/h, control vs. HF). The increase in aldosterone was accompanied by a 2-fold increase in adrenal aldosterone synthase mRNA expression and zona glomerulosa hypertrophy. Rats were also studied after 8 wk of HF diet, a time when MAP, but not body weight, was increased. At this time plasma aldosterone was unchanged but plasma renin activity was increased (4.4 ± 0.5 vs. 8.1 ± 1.3 ng/ml/h; control vs. HF, P < 0.05). These studies suggest that rats fed a HF diet from weaning may be a useful model for studying obesity-associated hyperaldosteronism.

Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

A custom rat and baboon hypertension gene array to compare experimental models.

One challenge in understanding the polygenic disease of hypertension is elucidating the genes involved and defining responses to environmental factors. Many studies focus on animal models of hypertension; however, this does not necessarily extrapolate to humans. Current technology and cost limitations are prohibitive in fully evaluating hypertension within humans. Thus, we have designed a single-array platform that allows direct comparison of genes relevant to hypertension in animal models and non-human primates/human hypertension. The custom array is targeted to 328 genes known to be potentially related to blood pressure control. Studies compared gene expression in the kidney from normotensive rats and baboons. We found 74 genes expressed in both the rat and baboon kidney, 41 genes expressed in the rat kidney that were not detected in the baboon kidney and 34 genes expressed in the baboon kidney that were not detected in the rat kidney. To begin the evaluation of the array in a pathological condition, kidney gene expression was compared between the salt-sensitive deoxycorticosterone acetate (DOCA) rat model of hypertension and sham animals. Gene expression in the renal cortex and medulla from hypertensive DOCA compared with sham rats revealed three genes differentially expressed in the renal cortex: annexin A1 (up-regulated; relative intensity: 1.316 ± 0.321 versus 2.312 ± 0.283), glutamate-cysteine ligase (down-regulated; relative intensity: 3.738 ± 0.174 versus 2.645 ± 0.364) and glutathione-S transferase (down-regulated; relative intensity: 5.572 ± 0.246 versus 4.215 ± 0.411) and 21 genes differentially expressed in the renal medulla. Interestingly, few genes were differentially expressed in the kidney in the DOCA-salt model of hypertension; this may suggest that the complexity of hypertension may be the result of only a few gene-by-environment responsive events.

Chronic estradiol-17ß exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: reversal by resveratrol.

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17ß (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.

Adenoviral inhibition of AT1a receptors in the paraventricular nucleus inhibits acute increases in mean arterial blood pressure in the rat.

Brain and peripheral renin-angiotensin systems are important in blood pressure maintenance. Circulating ANG II stimulates brain RAS to contribute to the increase mean arterial pressure (MAP). This mechanism has not been fully clarified, so it was hypothesized that reducing angiotensin type 1a (AT(1a)) receptors (AT(1a)Rs) in the paraventricular nucleus (PVN) would diminish intravenous ANG II-induced increases in MAP. Adenoviruses (Ad) encoding AT(1a) small hairpin RNA (shRNA) or Ad-LacZ (marker gene) were injected into the PVN [1 × 10(9) plaque-forming units/ml, bilateral (200 nl/site)] of male Sprague-Dawley rats instrumented with radiotelemetry transmitters for MAP and heart rate measurements and with venous catheters for drug administration. No differences in weight gain or basal MAP were observed. ANG II (30 ng·kg(-1)·min(-1) iv, 15 µl/min for 60 min) was administered 3, 7, 10, and 14 days after PVN Ad injection to increase blood pressure. ANG II-induced elevations in MAP were significantly reduced in PVN Ad-AT(1a) shRNA rats compared with Ad-LacZ rats (32 ± 6 vs. 8 ± 9 mmHg at 7 days, 35 ± 6 vs. 10 ± 6 mmHg at 10 days, and 32 ± 2 vs. 1 ± 5 mmHg at 14 days; P < 0.05). These observations were confirmed by acute administration of losartan (20 nmol/l, 100 nl/site) in the PVN prior to short-term infusion of ANG II; the ANG II-pressor response was attenuated by 69%. In contrast, PVN Ad-AT(1a) shRNA treatment did not influence phenylephrine-induced increases in blood pressure (30 µg·kg(-1)·min(-1) iv, 15 µl/min for 30 min). Importantly, PVN Ad-AT(1a) shRNA did not alter superior mesenteric arterial contractility to ANG II or norepinephrine; ACh-induced arterial relaxation was also unaltered. ß-Galactosidase staining revealed PVN Ad transduction, and Western blot analyses revealed significant reductions of PVN AT(1) protein. In conclusion, PVN-localized AT(1)Rs are critical for short-term circulating ANG II-mediated elevations of blood pressure. A sustained suppression of AT(1a)R expression by single administration of shRNA can interfere with short-term actions of ANG II.

Upregulated function of phosphatidylinositol-3-kinase in genetically hypertensive rats: a moderator of arterial hypercontractility.

1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals. 2. Aorta from spontaneously hypertensive rats (SHR; systolic blood pressure = 183 +/- 4 mmHg) and Wistar Kyoto (WKY) rats (115 +/- 2 mmHg) were isolated for the measurement of isometric contractile force. Aorta from SHR displayed small increases (approximately 5% maximum phenylephrine (PE)-induced contraction) in spontaneous tone, whereas aorta from WKY rats displayed none. The non-selective PI3K inhibitor LY294002 (20 micromol/L) and the selective inhibitor of the p110delta catalytic subunit of PI3K IC87114 (20 micromol/L) caused a fall of basal tone in SHR aorta (20 +/- 7 and 24 +/- 6% of the initial PE contraction, respectively), but did not alter tone in arteries from WKY rats. LY294002, but not IC87114, normalized the increased potency of noradrenaline (NA) observed in aorta from SHR (-log EC50 values for NA in the presence of vehicle in WKY rats and SHR 7.5 +/- 0.1 and 7.8 +/- 0.1, respectively (P < 0.05); -log EC(50) values for NA in the presence of LY294002 in WKY rats and SHR 7.0 +/- 0.1 and 7.0 +/- 0.1, respectively). 3. Biochemical expression of the p110 catalytic and p85 regulator subunits of PI3K in western analyses revealed no difference in expression of the regulatory p85alpha or p110alpha protein subunits between WKY rats and SHR; p110gamma was not detected. In contrast, p110delta expression was increased greater than 30% in aorta from SHR compared with WKY rats (827.6 +/- 88.5 vs 576.8 +/- 53.4 arbitrary densitometry units, respectively). Immunohistochemical analyses revealed expression of the p110delta isoform in the smooth muscle of arteries. 4. These data underscore the relevance of an enzyme historically classified as one committed to growth/anti-apoptosis in modifying contractility and supports involvement of PI3K in genetically based hypertension.

Effects of PPAR-gamma ligands on vascular smooth muscle marker expression in hypertensive and normal arteries.

Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant (P < 0.001) decrease in systolic blood pressure (BP; 149.9 +/- 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 +/- 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant (P < 0.001) decrease in BP (DOCA salt, 164.2 +/- 9.8 vs. DOCA-RS, 124.9 +/- 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 microM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-gamma ligand 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2); 20 microM) and TG (50 microM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ(2) and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 microM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-gamma agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.