letters of support). Cross-breeding with male (hREN)LtOJ TG rats produces (hAGT)L1623 TG dams that exhibit gestational hypertension. These rats will be used primarily for studies detailed in Project 5 (Brosnihan). PHS 398/2590 (Rev. 11/07) Page 437 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrarJO, CarlOS Maria SPECIFIC AIM 4 Core D will provide surgical and analytical expertise in the following procedures for all Projects. Drs. Strawn, Varagic, Diz and Shaltout have experience in these techniques;additional training will be provided by consultants. Ms Moore will serve as technical support for all surgical procedures and data analysis. 1. Long term measurement of intravascular pressure using telemetry devices and osmotic mini- Dump implantation: When experiments in this Program Project require rats prepared for chronic direct measurement of arterial pressure, animals will be instrumented with telemetry catheter transducers purchased from Data Sciences International. This method of blood pressure recording has two distinct advantages. First, it facilitates the continuous monitoring of blood pressure for periods of time up to six months. The rats are housed individually and minimally impacted since they have been instrumented with a catheter transducer that transmits blood pressure signals to a receiver unit below the rat cage. Second, the continuous measurement of blood pressure permits the reliable detection of small changes in blood pressure (~5 mm Hg). Further, since animals are chronically instrumented with a telemetry transducer, this method of blood pressure measurement facilitates experimental deigns that take advantage of statistical analyses utilizing repeated measurements ANOVA. Thus, one can assess the blood pressure before and after experimental manipulations such as drug or dietary treatments (Projects 1 and 5) or induction of pregnancy (Project 5). Publications and experiments using telemetry devices are detailed in Projects 1 and 5. To prepare animals for the telemetry measurement of blood pressure, rats or mice are anesthetized with ketamine/acepromazine (40-80/2.5 mg/kg) or isoflurane (1-2%) as appropriate with the aims of the particular experiment. The animal is prepared for aseptic surgery, shaved, and a ventral midline incision is made in the abdomen. The intestines are displaced to facilitate exposure of the abdominal aorta. A section of the aorta 1-2 cm proximal to the terminal aortic bifurcation (in the case of rats) is separated from the vena cava. A small hole is made in the aorta with a 23 gauge needle and the telemetry catheter (rats: TA11PA-C40; mice: TA11PA-C20) is inserted into the vessel. A small piece of Dacron"""""""" material is inserted between the external portion of the catheter and the aorta;Vetbond"""""""" (biocompatible super glue) is applied to the incision site to help anchor the catheter to the vessel. The other end of the catheter which is connected to the transducer and telemetry battery pack is sutured to the abdominal wall as the abdominal incision is closed. The abdominal muscle layers are approximated with interrupted sutures and the skin incision is then closed. The animals are treated post-operatively with penicillin G (30,000 units) and an analgesic (buprenorphine;0.1- 0.5 mg/kg). The health of animals with a telemetry catheter is checked regularly to insure that the abdominal incision is healing well. Rats and mice are permitted to recover for a minimum of 10 days before collection of blood pressure data is begun. Our experience in rats has shown that regular diurnal fluctuations in blood pressure and heart rate do not return until 10-12 days after implantation of the telemetry catheter. The telemetry system that will be used includes a data acquisition interface so that continuously acquired blood pressure and heart rate as well as animal activity is stored on a computer. The data acquisition system provided by Data Sciences International permits the analysis of blood pressure and heart rate over user defined intervals. Thus, it is convenient to monitor the diurnal variation of blood pressure and heart rate. Osmotic mini-pumps will be used in all 5 projects of the Program. Osmotic minipumps (ALZET models) will be implanted for subcutaneous, intraperitoneal, intracerebroventricular, intrauterine, and intravenous infusion. In all cases, the surgical implantation site will be prepared by aseptic techniques in rats anesthetized with ketamine/acepromazine (40-80/2.5 mg/kg) or isoflurane (1-2%). For subcutaneous and intravenous infusions, a small (1 cm) incision will be made in the skin overlying the area between scapulae and a pre-loaded pump will be inserted through the incision into a subcutaneous pocket prepared by blunt dissection. When intravenous infusion accompanies the subcutaeous implantation, appropriate size catheter tubing will be attached to the pump portal after guidance and fixation into a jugular vein or lateral ventricle. Catheterization will be performed through a small (1 cm) skin incision overlying the jugular vein;approximately 2 cm of tubing will be fixed within the vein, and the remainder will be tunneled under the skin for attachment to the pump portal. Lateral ventricle cannulae will be placed as described in Project 4.Skin will be closed with 3-0 non-absorbable suture. Intraperitoneal placement of pumps will be performed through skin and abdominal wall incisions (1 cm each). Both incisions will be closed individually with 3-0 non-absorbable suture. 2. Long-term Measurement of Cardiac Output or Regional Blood Flow: Ascending aortic or regional blood flows will be measured by implantation of flow probes manufactured by Transonic Systems, Inc. This type of flowmeter system measures blood flow by ultrasonic transit-time technology and, thereby, provides a direct measurement of volume flow in ml/min. Chronic measurement of cardiac output will made in PHS 398/2590 (Rev. 11/07) Page 438 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, CarlOS Maria experiments of Project 5 that focus on systemic hemodynamic regulation during pregnancy. Rats will be anesthetized with ketamine/acepromazine (40-80/2.5 mg/kg) or halothane (1-2%) as appropriate with the aims of the particular experiment, prepared for aseptic surgery, and the trachea intubated. The rat is then placed on a ventilator and ventilated at a rate of 90 breaths per minute and a tidal volume of 1-2 ml per stroke. An incision is made in the right thoracic wall and the thoracic cavity opened via the third intercostal space. The right lung is compressed with a neurosurgical sponge and the ascending aorta visualized. Adventitial tissue is dissected away from the ascending aorta. A flow probe (Transonic Systems Inc.;2PSB) is placed around the ascending aorta and the electrical cable from the flow probe brought out through the second intercostal space. The neurosurgical sponge is removed, the lungs reinflated, and thoracic cavity closed. The thorax is evacuated via a chest tube introduced through the sixth intercostal space. The cable of the flow probe is tunneled subcutaneously, brought out through the skin button between the shoulder blades, and led through the stainless steel spring. When the above surgical procedures have been completed, the rat will be treated post-operatively with penicillin G (30,000 units). The animal is then placed in its home cage, the leads from the flow probe connected to an electrical commutator (Dragonfly, Inc.;Model SL-36). The electrical commutator is connected to a flowmeter (Transonic Systems Inc.;Model TS420). In some experiments of Project 5 rats will also be instrumented for the chronic measurement of blood pressure as well as ascending aortic blood flow. With this type of preparation, Dr. Brosnihan will be able to monitor changes in total peripheral resistance in addition to alterations in blood pressure and cardiac output during the course of pregnancy. In other experiments blood flow to the mesenteric), hindlimb, or renal circulations will be made. In these experiments, rats will be anesthetized as described above and the vessel of interest exposed (mesenteric artery, terminal aorta, or main renal artery). In most cases a 1 mm flow probe (Transonic Systems, Inc.;1PRB) will be used for measurement of blood flow. 3. Assessment of Left Ventricular Structure and Function by Transthoracic Echocardioaraphv (TTE): TTE examinations is conducted on rats anesthetized with ketamine/xylazine (50mg/kg/5mg/kg;i.m.) using a commercially available sector scanner equipped with a 12 MHz phased-array transducer (Philips Medical Systems,Andover, MA). Animals are place in a left lateral supine position and the TTE probe is placed carefully to obtain the short and long axis as well as 4 -chamber apical cardiac views. From the short axis, an M-mode tracing of the left ventricle (LV) is obtained and measurement of LV end-diastolic diameter (LVDD), LV systolic diameter (LVSD), posterior (PWT), and septum (SWT) diastolic wall thicknesses is made according to the American Society of Echocardiography guidelines (1).The percentage of LV fractional shortening (FS), an index of global systolic function, is calculated as ((LVDD - LVSD)/LVDD) x 100.To asses diastolic function, mitral inflow measurements of early and late filling velocities (E and A, respectively), deceleration slope (Edec slope), and early deceleration time (Edec time) are obtained using pulsed Doppler, with the sample volume placed at the tips of mitral leaflets from an apical four-chamber orientation. Doppler tissue imaging to assess early mitral valve septal annular velocities (e1) is also obtained from the four-chamber view. This parameter is believed to be less influenced by ventricular load conditions. Ratio of E/e',an index of LV filling pressure, is also calculated. All measured and calculated systolic and diastolic indices are represented as the average of at least five consecutive cardiac cycles to minimize beat-to-beat variability. Representative examples are provided in Figure 1. Figure 1. Representative echocardiographic images. Left Panel: M-mode echocardiogram with end-diastolic and end-systolic dimensions shown;Middle Panel: Pulse-wave Doppler spectra (E and A velocity) of mitral inflow;Right Panel: Tissue Doppler imaging illustrating myocardial tissue velocity at the septal mitral annular region. Figures are from experiments of Project 1 in (mRen2)27 rats (Left, Right) and an SHR (Middle). PHS 398/2590 (Rev.11/07) Page 439 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FerrariO, Carlos Maria We have several paper published in which we used echocardiographic and Doppler techniques for the assessment of left ventricular structure and function (2-7). 4. Analysis of Central Regulation of Blood Pressure by Evaluation of Spontaneous Baroreflex Sensitivity. Blood pressure and heart rate will be measured directly through an indwelling catheter in the femoral artery or telemetry probe situated in the aorta. Systolic arterial pressure (SAP) and RR interval (RRI) files generated via the BIOPAC acquisition software (Santa Barbara, CA) or Datasciences telemetry at 1000 HZ will be analyzed using Nevrokard SA-BRS software (Nevrokard SA-BRS, Medistar, Ljubljana, Slovenia) through the following methods. a. Frequency Method: Power spectral densities of SAP and RRI oscillations will be computed by 512 points Fast Fourier Transform (FFT) and integrated over the specified frequency range (LF;0.25-0.75 Hz) and (HF;0.75-5.0 Hz). A Hanning window will be applied and the spectra of SAP and RRI series, and their squared-coherence modulus, will be computed if the coherence is greater than 0.5 in accordance with reported criteria (8). The square-root of the ratio of RRI's and SAP powers will be computed to calculate LF, HF alpha indices, which reflect the baroreflex sensitivity. Power of RRI spectra in LF, HF range (LFRR| and HFRR|) will be calculated in normalized units and the ratio of LFRR|/HFRR| will be used as a measure of sympathovagal balance (9). Power of SAP spectra is calculated as LFSAp will be used as a measure of blood pressure variability (BPV). b. Sequence Method: Spontaneous baroreflex sensitivity calculated by this method is based on quantification of sequences of at least three beats (n) in which SAP consecutively increases (UP sequence) or decreases (DOWN sequence), accompanied by changes in the same direction of the RRI of the subsequent beats (n+1). In order to be included in the baroreflex sensitivity estimate, each sequence must fulfill the following criteria(10): (1) minimal RRI change 3 ms;(2) minimal SAP change 1 mm Hg;(3) minimal number of beats, 3 or more, in the sequence;(4) minimal correlation coefficient of 0.85. The software scans the RRI and SAP records, identifies sequences, and then calculates linear correlation between RRI and SAP for each sequence. If the correlation coefficient exceeds a pre-set critical value (0.85), the regression coefficient (slope) is calculated and accepted. The mean of all individual regression coefficients (slopes), which is a measure of sequence BRS is then calculated. Overall, three parameters will be obtained by this method (Sequence BRS- SAP UP, DOWN and TOTAL). c. Time-Domain Analysis: Three time-domain SDRR rMSSD parameters will be used to measure C pre hemodynamic variability as in previous studies (11;12). Heart rate variability (HRV) is determined by computing the standard deviation of beat-to-beat interval (SDRR) and the C pre C post 1-7 pre 1-7 post C pro C post 1-7 pre 1-7 post root mean square of successive beat-to-beat differences in R-R interval duration (rMSSD). The Seq All standard deviation of the mean arterial pressure (SDMAP) is used as a measure for blood pressure variability (BPV). The Nevrokard software has been used in studies in sheep, rat Cpra Cpost 1-7 pre 1-7 post Cpra Cpost 1-7 pre 1-7post and humans and has been validated by us against classical measures of Figure 2. C pre (white bar), C post (gray bars) correspond to parameters before and baroreflex function in sheep and rats after intracisternal injection of the control fusion protein, whereas 1-7 pre (black bar) (13-15). The software allows and 1-7 post (gray bar) correspond to before and after intracisternal injection of Ang- analysis of blood pressure data (1-7) fusion protein. acquired by Biopac, Data Science and a variety of other commercially available systems. Samples of each of the measures in rats from data in PHS 398/2590 (Rev. 11/07) Page 440 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FeiTariO, CarlOS Maria Project 4 are shown in Figure 2. Results indicate that intercisternal injection of angiotensin-(1-7) increases SDRR, rMSSD, HF indices compared to injection of a control fusion protein (CTL-FP) or prior to ang-(1-7) FP injection. Figure 3. Representative images and tracings from the uterine artery of a pregnant rat.showinq blood flow velocity. Figure 4. Representative example of ultrasound determination of uterine artery size and blood flow. PHS 398/2590 (Rev.11/07) Page 441 5. Ultrasound Dopp/er Flow. Studies using this technique are detailed in the experiments of Project 5 (Brosnihan). Pregnant animals will be anesthetized with isoflurane and positioned on a rat handling platform. All of the hair will be removed from the abdomen and pre-warmed gel will be used as a coupling medium. Maternal heart rates and rectal temperatures will be monitored. A Vevo 770"""""""" High- Resolution in vivo Imaging System (VisualSonics, Inc, Toronto, Canada) is available in the laboratory of Dr. April Ronca (see letter of consultation for Project 5). Pregnant rats will be imaged with a RMV ? 71 OB High Frame Rate Scanhead (Real-time Micro Visualization) probe and a PW (Pulsed Wave Doppler) transducer. The high- pass filter will be set at 6 Hz, and the pulsed repetition frequency will be set at 20 kHz. A 0.2 -0.5 mm pulsed Doppler gate will be used and the angle between the Doppler beam and the vessel will be set at <30?. Doppler waveforms will be obtained from the proximal and distal uterine artery and the arcuate artery. Peak systolic velocity (PSV) and end-diastolic velocity (EDV) will be measured from 3 consecutive cardiac cycles that are not affected by maternal breathing. The resistance index will be calculated (RI=[PSV-EDV]/PSV. The flow in placenta bed veins will also be monitored. Figures 3 and 4 illustrate the capacity of this technique to evaluate uterine artery blood flow and size. Fig. 4 specifically shows the placenta anatomy of Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FeiTariO, Carlos Maria a pregnant rat at day 20 of gestation using ultrasound. The maternal placenta (mesometrial triangle) and fetal placenta (labyrinth) are shown. A blood vessel (white arrow) that traverses the fetal placenta is also present, from which vessel diameter and flow can be determined. Progress Report A subset of (mRen2)27 TG rats that exhibit heritable malignant hypertension was identified during the final years of the previous grant cycle;we are currently in the process of creating a colony of these rats through selective breeding and offspring phenotyping techniques under separate funding. A subset of Hann SD females was identified that that when bred with homozygote (mRen2)27 males allow increased hemizygote offspring expression of the malignant hypertensive phenotype. This subset of (mRen2)27 TG rats develop malignant hypertension at about 8 weeks of age;these animals generally succumb to stroke, aneurysm, or acute heart/renal failure. Expression of the phenotype is related to the Hann SD background, since previous reports by Whitworth et al. (16)showed that the phenotype was eliminated or intensified depending on strain background. Currently, there are no specified experiments or funds required within the grant application for use of this model;we believe, however, that this is an important model that may provide future experimental applications. National Significance of the Breeding Colonies The success of the Transgenic Animal Colony to provide animals for significant research efforts is reflected by the number of publications in which Colony animals were used (see individual Projects for these references). In addition, the importance of the Colony as a national resource for transgenic rats is demonstrated by the fact that approximately 50% of rats produced in Grant Years 13 and 14 for research purposes were shipped to outside investigators at cost. The Lew.mRen2 congenic rat colony is in production and remains the sole source of this unique hypertensive rat model;several publications resulted from use of this new genetic model during the grant year;production of this model represents an increasing percentage of total production. Finally, the identification of a subset of (mRen2)27 with heritable malignant hypertension and establishment of a colony with the phenotype will provide the single world-wide source of this model of hypertension. Table 1 lists the number of animals by colony provided to investigators of the PPG as well as to collaborating investigators residing outside Wake Forest University [Drs. Aileru (Winston-Salem State University), Clark (Nova Southeastern University, Fort Lauderdale, FL), Sowers (Harry S. Truman VA Hospital, Columbia, MO), Whaley-Connell (University of Missouri-Columbia), Henriksen (University of Arizona, Tucson), Villareal (University of California, San Diego]. Yr11 Yr12 Yr13 Yr14 Hann SD 154 139 333 273 (mRen2)27 301 157 497 413 mRen2.Lewis 109 117 224 258 ASrAogen 79 117 108 9 ACE2-KO 0 0 0 86 Totals 643 400 1162 1039 Table 1. Number of animals produced by Core D for distribution to Projects and outside investigators. Publications The Hypertension Center Transgenic Animal Core has provided animals for all of the projects of thePPG. Publications using animals from the Core are found within the individual projects. References 1. Sahn.D.J., DeMaria.A., Kisslo.J., and Weyman.A. 1978. Recommendations regarding quantitation in M- mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58:1072- 1083. PHS 398/2590 (Rev. 11/07) Page 442 Continuation Format Page Program Director/Principal Investigator (Last, First, Middle): FeiTariO, CarlOS Maria 2. Ahn.J., Varagic.J., Slama.M., Susic.D., and Frohlich.E.D. 2004. Cardiac structural and functional responses to salt loading in SHR. Am J Physiol Heart Circ Physiol 287:H767-H772. 3. Groban.L, Yamaleyeva.LM., Westwood.B.M., Houle.T.T., Lin,M., Kitzman.D.W., and Chappell.M.C. 2008. Progressive diastolic dysfunction in the female mRen(2).Lewis rat: influence of salt and ovarian hormones. J Gerontol A Biol Sci Med Sci 63:3-11. 4. Slama,M., Ahn.J., Varagic.J., Susic.D., and Frohlich.E.D. 2004. Long-term left ventricular echocardiographic follow-up of SHR and WKY rats: effects of hypertension and age. Am J Physiol Heart Circ Physiol 286:H181-H185. 5. Slama.M., Ahn.J., Peltier.M., Maizel.J., Chemla.D., Varagic.J., Susic.D., Tribouilloy.C., and Frohlich.E.D. 2005. Validation of echocardiographic and Doppler indexes of left ventricular relaxation in adult hypertensive and normotensive rats. Am J Physiol Heart Circ Physiol 289:H1131-H1136. 6. Varagic.J., Frohlich.E.D., Diez.J., Susic.D., Ahn.J., Gonzalez.A., and Lopez.B. 2006. Myocardial fibrosis, impaired coronary hemodynamics, and biventricular dysfunction in salt-loaded SHR. Am J Physiol Heart Circ Physiol 290:H1503-H1509. 7. Varagic.J., Frohlich.E.D., Susic.D., Ahn.J., Matavelli.L., Lopez.B., and Diez.J. 2008. AT1 receptor antagonism attenuates target organ effects of salt excess in SHRs without affecting pressure. Am J Physiol Heart Circ Physiol 294:H853-H858. 8. Parati.G., Frattola.A., Di Rienzo.M., Castiglioni.P., Pedotti.A., and Mancia.G. 1995.Effects of aging on 24-h dynamic baroreceptor control of heart rate in ambulant subjects. Am J Physiol 268:H1606-H1612. 9. Laitinen.T., Hartikainen.J., Niskanen.L, Geelen.G., and Lansimies.E. 1999.Sympathovagal balance is major determinant of short-term blood pressure variability in healthy subjects. Am J Physiol 276:H1245- H1252. 10. Wang,Y.P., Cheng,Y.J., and Huang,C.L. 2004. Spontaneous baroreflex measurement in the assessment of cardiac vagal control. Clin Auton Res 14:189-193. 11. Sgoifo.A., de Boer.S.F., Westenbroek.C., Maes.F.W., Beldhuis.H., Suzuki,!., and Koolhaas.J.M. 1997. Incidence of arrhythmias and heart rate variability in wild-type rats exposed to social stress. Am J Physiol 273:H1754-H1760. 12. Stein,P.K., Bosner,M.S., Kleiger.R.E., and Conger.B.M. 1994. Heart rate variability: a measure of cardiac autonomic tone. Am Heart J127:1376-1381. 13. Shaltout.HA, Figueroa.J.P., Rose.J.C., Chappell.M.C., Diz.D.I., and Averill.D.B. 2006. Antenatal betamethasone causes angiotensin ll-mediated impairment of baroreflex control of heart rate. Journal of Hypertension 24:159. 14. Shaltout.H.A., and bdel-Rahman,A.A. 2005. Mechanism of fatty acids induced suppression of cardiovascular reflexes in rats. J Pnarmacol Exp 77)er314:1328-1337. 15. Hughes,J.W., Dennis.M.F., and Beckham.J.C. 2007. Baroreceptor sensitivity at rest and during stress in women with posttraumatic stress disorder or major depressive disorder. J Trauma Stress 20:667-676. 16. Whitworth CE, Fleming S, Kotelevtsev Y, Manson L, Brooker GA, Gumming AD, Mullins JJ. A genetic model of malignant phase hypertension in rats. Kidney Int 1995;47:529-35. PHS 398/2590 (Rev. 11/07) Page 443 Continuation Format Page Program Director/Principal Investigator(Last, First, Middle): FerrarJO, Carlos Maria RESOURCES FACILITIES: Specify the facilities to be used for the conductof the proposed research. Indicate the project/

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Dell'Italia, Louis J; Collawn, James F; Ferrario, Carlos M (2018) Multifunctional Role of Chymase in Acute and Chronic Tissue Injury and Remodeling. Circ Res 122:319-336
Ahmad, Sarfaraz; Ferrario, Carlos M (2018) Chymase inhibitors for the treatment of cardiac diseases: a patent review (2010-2018). Expert Opin Ther Pat 28:755-764
Wang, Hao; Sun, Xuming; Lin, Marina S et al. (2018) G protein-coupled estrogen receptor (GPER) deficiency induces cardiac remodeling through oxidative stress. Transl Res 199:39-51
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Li, Tiankai; Zhang, Xiaowei; Cheng, Heng-Jie et al. (2018) Critical role of the chymase/angiotensin-(1-12) axis in modulating cardiomyocyte contractility. Int J Cardiol 264:137-144
Chappell, Mark C; Al Zayadneh, Ebaa M (2017) Angiotensin-(1-7) and the Regulation of Anti-Fibrotic Signaling Pathways. J Cell Signal 2:
Alencar, Allan K; da Silva, Jaqueline S; Lin, Marina et al. (2017) Effect of Age, Estrogen Status, and Late-Life GPER Activation on Cardiac Structure and Function in the Fischer344√óBrown Norway Female Rat. J Gerontol A Biol Sci Med Sci 72:152-162
Guichard, Jason L; Rogowski, Michael; Agnetti, Giulio et al. (2017) Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure. Am J Physiol Heart Circ Physiol 313:H32-H45
Brosnihan, K Bridget; Chappell, Mark C (2017) Measurement of Angiotensin Peptides: HPLC-RIA. Methods Mol Biol 1527:81-99
Butts, Brittany; Goeddel, Lee A; George, David J et al. (2017) Increased Inflammation in Pericardial Fluid Persists 48 Hours After Cardiac Surgery. Circulation 136:2284-2286

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